experimental-melanoma-immunotherapy-model-using-tumor-vaccination

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been ...

La Jolla Institute for Immunology

Protein kinase C-θ (PKCθ) is an important component of proximal T cell receptor (TCR) signaling. We previously identified the amino-terminal C2 domain of PKCθ as a phosphotyrosine (pTyr)-binding domain. Using a mutant form of PKCθ that cannot bind pTyr (PKCθ), we showed that pTyr binding by PKCθ was required for TCR-induced T cell activation, proliferation, and T2 cell differentiation but not for T cell development. Using tandem mass spectrometry and coimmunoprecipitation, we identified the kinase ζ-associated protein kinase of 70 kDa (Zap70) as a binding partner of the PKCθ pTyr-binding pocket. Tyr of Zap70 directly bound to PKCθ, and the interdomain B residues Tyr and Tyr were indirectly required for binding to PKCθ, reflecting their role in promoting the open conformation of Zap70. PKCθ-expressing CD4 T cells displayed defects not only in known PKCθ-dependent signaling events, such as nuclear factor κB (NF-κB) activation and T2 cell differentiation, but also in full activation of Zap70 itself and in the activating phosphorylation of linker of activation of T cells (LAT) and phospholipase C-γ1 (PLCγ1), signaling proteins that are traditionally considered to be activated independently of PKC. These findings demonstrate that PKCθ plays an important role in a positive feedback regulatory loop that modulates TCR-proximal signaling and, moreover, provide a mechanistic explanation for earlier reports that documented an important role for PKCθ in T cell Ca signaling. This PKCθ-Zap70 interaction could potentially serve as a promising and highly selective immunosuppressive drug target in autoimmunity and organ transplantation.

Phosphotyrosine-dependent interaction between the kinases PKCθ and Zap70 promotes proximal TCR signaling.

We reported that protein kinase C-η (PKCη) forms a novel (to our knowledge) signaling complex with the checkpoint inhibitory protein CTLA-4 in regulatory T cells (Tregs). This complex is required for the contact-dependent suppressive activity of Tregs, including suppression of antitumor immunity. However, the importance of PKCη in protective immunity mediated by T effector cells remains unclear. We used mice with germline or conditional Treg-specific deletion of , the PKCη-encoding gene, to explore CD8 T cell-dependent antiviral immunity using the lymphocytic choriomeningitis virus Armstrong strain acute infection model as well as the in vitro activation of murine or human CD8 T cells. Five days following infection, germline   mice displayed enhanced viral clearance compared with control mice. Similarly,  Treg-specific conditional knockout mice also showed improved viral clearance and displayed enhanced expression of granzyme B and IFN-γ by both virus-specific and total CD8 T cells, demonstrating that enhanced viral clearance in germline   mice is caused by PKCη deficiency in Tregs and the resulting functional defect of   Tregs. In addition, purified   mouse CD8 T cells as well as  knockdown human CD8 T cells displayed intact, or even enhanced, T cell activation in vitro as measured by proliferation and expression of granzyme B and IFN-γ. Thus, global PKCη deletion does not impair overall CD8 T cell-mediated immunity, including antiviral immunity, implying that selective pharmacological PKCη inhibition could be safely used in vivo to inhibit undesired contact-dependent suppression by Tregs and, thus, enhance tumor-specific and, likely, virus-specific immunity.

Protein Kinase C-η Deficiency Does Not Impair Antiviral Immunity and CD8 T Cell Activation.

Our previous studies revealed a critical role of a novel CTLA4-protein kinase C-eta (PKCη) signaling axis in mediating the suppressive activity of regulatory T cells (Tregs) in antitumor immunity. These studies have employed adoptive transfer of germline PKCη-deficient ( ) Tregs into   mice prior to tumor implantation. Here, we extended these findings into a biologically and clinically more relevant context.

Ann J. Canonigo-Balancio

La Jolla Institute for Immunology

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine