Protein Kinase C Theta Modulates PCMT1 through hnRNPL to Regulate FOXP3 Stability in Regulatory T Cells.

T cell receptor signaling, together with cytokine-induced signals, can differentially regulate RNA processing to influence T helper versus regulatory T cell fate. Protein kinase C family members have been shown to function in alternative splicing and RNA processing in various cell types. T cell-specific protein kinase C theta, a molecular regulator of T cell receptor downstream signaling, has been shown to phosphorylate splicing factors and affect post-transcriptional control of T cell gene expression. In this study, we explored how using a synthetic cell-penetrating peptide mimic for intracellular anti-protein kinase C theta delivery fine-tunes differentiation of induced regulatory T cells through its differential effects on RNA processing. We identified protein kinase C theta signaling as a critical modulator of two key RNA regulatory factors, heterogeneous nuclear ribonucleoprotein L (hnRNPL) and protein-l-isoaspartate O-methyltransferase-1 (PCMT1), and loss of protein kinase C theta function initiated a "switch" in post-transcriptional organization in induced regulatory T cells. More interestingly, we discovered that protein-l-isoaspartate O- methyltransferase-1 acts as an instability factor in induced regulatory T cells, by methylating the forkhead box P3 (FOXP3) promoter. Targeting protein-l-isoaspartate O-methyltransferase-1 using a cell-penetrating antibody revealed an efficient means of modulating RNA processing to confer a stable regulatory T cell phenotype.

Cell-Penetrating Anti-Protein Kinase C Theta Antibodies Act Intracellularly to Generate Stable, Highly Suppressive Regulatory T Cells.

Regulatory T cells maintain immunological tolerance and dampen inflammatory responses. Administering regulatory T cells can prevent the immune-mediated tissue destruction of graft-versus-host disease, which frequently accompanies hematopoietic stem cell transfer. Neutralizing the T cell-specific kinase, protein kinase C theta, which promotes T cell effector functions and represses regulatory T cell differentiation, augments regulatory T cell immunosuppression and stability. We used a synthetic, cell-penetrating peptide mimic to deliver antibodies recognizing protein kinase C theta into primary human CD4 T cells. When differentiated ex vivo into induced regulatory T cells, treated cells expressed elevated levels of the regulatory T cell transcriptional regulator forkhead box P3, the surface-bound immune checkpoint receptor programmed death receptor-1, and pro-inflammatory interferon gamma, previously ascribed to a specific population of stable, highly suppressive human induced regulatory T cells. The in vitro suppressive capacity of these induced regulatory T cells was 10-fold greater than that of T cells differentiated without antibody delivery. When administered at the time of graft-versus-host disease induction, using a humanized mouse model, antibody-treated regulatory T cells were superior to non-treated T cells in attenuating lethal outcomes. This antibody delivery approach may overcome obstacles currently encountered using patient-derived regulatory T cells as a cell-based therapy for immune modulation.

CD28 Signaling Drives Notch Ligand Expression on CD4 T Cells.

Notch signaling provides an important cue in the mammalian developmental process. It is a key player in T cell development and function. Notch ligands such as Delta-like ligands (DLL) 1, 3, 4, and JAG1, 2 can impact Notch signaling positively or negatively, by trans-activation or cis-inhibition. Trans and cis interactions are receptor-ligand interaction on two adjacent cells and interaction on the same cell, respectively. The former sends an activation signal and the later, a signal for inhibition of Notch. However, earlier reports suggested that Notch is activated in the absence of Notch ligand-expressing APCs in a purified population of CD4 T cells. Thus, the role of ligands in Notch activation, in a purified population of CD4 T cells, remains obscure. In this study, we demonstrate that mature CD4 T cells are capable of expressing Notch ligands on their surface very early upon activation with soluble antibodies against CD3 and CD28. Moreover, signaling solely through CD28 induces Notch ligand expression and CD3 signaling inhibits ligand expression, in contrast to Notch which is induced by CD3 signaling. Additionally, by using decoys, mimicking the Notch extracellular domain, we demonstrated that DLL1, DLL4, and JAG1, expressed on the T cells, can cis-interact with the Notch receptor and inhibit activation of Notch. Thus, our data indicate a novel mechanism of the regulation of Notch ligand expression on CD4 T cells and its impact on activated Notch.

Cymerus™ iPSC-MSCs significantly prolong survival in a pre-clinical, humanized mouse model of Graft-vs-host disease.

The immune-mediated tissue destruction of graft-vs-host disease (GvHD) remains a major barrier to greater use of hematopoietic stem cell transplantation (HSCT). Mesenchymal stem cells (MSCs) have intrinsic immunosuppressive qualities and are being actively investigated as a therapeutic strategy for treating GvHD. We characterized Cymerus™ MSCs, which are derived from adult, induced pluripotent stem cells (iPSCs), and show they display surface markers and tri-lineage differentiation consistent with MSCs isolated from bone marrow (BM). Administering iPSC-MSCs altered phosphorylation and cellular localization of the T cell-specific kinase, Protein Kinase C theta (PKCθ), attenuated disease severity, and prolonged survival in a humanized mouse model of GvHD. Finally, we evaluated a constellation of pro-inflammatory molecules on circulating PBMCs that correlated closely with disease progression and which may serve as biomarkers to monitor therapeutic response. Altogether, our data suggest Cymerus iPSC-MSCs offer the potential for an off-the-shelf, cell-based therapy to treat GvHD.

Intracellular Delivery of Anti-pPKCθ (Thr538) via Protein Transduction Domain Mimics for Immunomodulation.

Targeting cellular proteins with antibodies, to better understand cellular signaling pathways in the context of disease modulation, is a fast-growing area of investigation. Humanized antibodies are increasingly gaining attention for their therapeutic potential, but the collection of cellular targets is limited to those secreted from cells or expressed on the cell surface. This approach leaves a wealth of intracellular proteins unexplored as putative targets for antibody binding. Protein kinase Cθ (PKCθ) is essential to T cell activation, proliferation, and differentiation, and its phosphorylation at specific residues is required for its activity. Here we report on the design, synthesis, and characterization of a protein transduction domain mimic capable of efficiently delivering an antibody against phosphorylated PKCθ (Thr538) into human peripheral mononuclear blood cells and altering expression of downstream indicators of T cell activation and differentiation. We used a humanized, lymphocyte transfer model of graft-versus-host disease, to evaluate the durability of protein transduction domain mimic:Anti-pPKCθ modulation, when delivered into human peripheral mononuclear blood cells ex vivo. We demonstrate that protein transduction domain mimic:Antibody complexes can be readily introduced with high efficacy into hard-to-transfect human peripheral mononuclear blood cells, eliciting a biological response sufficient to alter disease progression. Thus, protein transduction domain mimic:Antibody delivery may represent an efficient ex vivo approach to manipulating cellular responses by targeting intracellular proteins.

Development of Guanidinium-Rich Protein Mimics for Efficient siRNA Delivery into Human T Cells.

RNA interference is gaining attention as a means to explore new molecular pathways and for its potential as a therapeutic; however, its application in immortal and primary T cells is limited due to challenges with efficient delivery in these cell types. Herein, we report the development of guanidinium-rich protein transduction domain mimics (PTDMs) based on a ring-opening metathesis polymerization scaffold that delivers siRNA into Jurkat T cells and human peripheral blood mononuclear cells (hPBMCs). Homopolymer and block copolymer PTDMs with varying numbers of guanidinium moieties were designed and tested to assess the effect cationic charge content and the addition of a segregated, hydrophobic block had on siRNA internalization and delivery. Internalization of fluorescently labeled siRNA into Jurkat T cells illustrates that the optimal cationic charge content, 40 charges per polymer, leads to higher efficiencies, with block copolymers outperforming their homopolymer counterparts. PTDMs also outperformed commercial reagents commonly used for siRNA delivery applications. Select PTDM candidates were further screened to assess the role the PTDM structure has on the delivery of biologically active siRNA into primary cells. Specifically, siRNA to hNOTCH1 was delivered to hPBMCs enabling 50-80% knockdown efficiencies, with longer PTDMs showing improved protein reduction. By evaluating the PTDM design parameters for siRNA delivery, more efficient PTDMs were discovered that improved delivery and gene (NOTCH) knockdown in T cells. Given the robust delivery of siRNA by these novel PTDMs, their development should aid in the exploration of T cell molecular pathways leading eventually to new therapeutics.