Loss-of-function phenotype of a PKCθT219A knockin mouse strain.

BACKGROUND: Protein kinase C θ has been established as an important signaling intermediate in T-effector-cell activation and survival pathways by controlling activity of the key transcription factors NF-κB and NFAT. Previous studies identified an activation-induced auto-phosphorylation site at Thr-219, located between the tandem C1 domains of the regulatory fragment in PKCθ, as a structural requirement for its correct membrane translocation and the subsequent transactivation of downstream signals leading to IL-2 production in a human T cell line. METHODS: The present work aimed to define the role of this phosphorylation switch on PKCθ in a physiological context through a homozygous T219A knockin mouse strain. T cell activation was analyzed by H3-thymidine uptake (proliferative response), qRT-PCR and luminex measurements (cytokine production). NFAT and NF-κB transactivation responses were estimated by Gel mobility shift and Alpha Screen assays. Frequencies of T cell subsets were analyzed by flow cytometry. RESULTS: Despite a normal T cell development, in vitro activated effector T cells clearly revealed a requirement of Thr-219 phosphorylation site on PKCθ for a transactivation of NF-κB and NFAT transcription factors and, subsequently, robust IL-2 and IFN-γ expression. CONCLUSION: This phenotype is reminiscent of the PKCθ knockout T cells, physiologically validating that this (p) Thr-219 auto-phosphorylation site indeed critically regulates PKCθ function in primary mouse T cells.

Prediction and validation of murine MHC class I epitopes of the recombinant virus VSV-GP.

Oncolytic viruses are currently tested as a novel platform for cancer therapy. These viruses preferentially replicate in and kill malignant cells. Due to their microbial origin, treatment with oncolytic viruses naturally results in anti-viral responses and general immune activation. Consequently, the oncolytic virus treatment also induces anti-viral T cells. Since these can constitute the dominant activated T cell pool, monitoring of the anti-viral T cell response may aid in better understanding of the immune responses post oncolytic virotherapy. This study aimed to identify the anti-viral T cells raised by VSV-GP virotherapy in C57BL/6J mice, one of the most widely used models for preclinical studies. VSV-GP is a novel oncolytic agent that recently entered a clinical phase I study. To identify the VSV-GP epitopes to which mouse anti-viral T cells react, we used a multilevel adapted bioinformatics viral epitope prediction approach based on the tools netMHCpan, MHCflurry and netMHCstabPan, which are commonly used in neoepitope identification. Predicted viral epitopes were ranked based on consensus binding strength categories, predicted stability, and dissimilarity to the mouse proteome. The top ranked epitopes were selected and included in the peptide candidate matrix in order to use a matrix deconvolution approach. Using ELISpot, we showed which viral epitopes presented on C57BL/6J mouse MHC-I alleles H2-Db and H2-Kb trigger IFN-γ secretion due to T cell activation. Furthermore, we validated these findings using an intracellular cytokine staining. Collectively, identification of the VSV-GP T cell epitopes enables monitoring of the full range of anti-viral T cell responses upon VSV-GP virotherapy in future studies with preclinical mouse models to more comprehensively delineate anti-viral from anti-tumor T cell responses. These findings also support the development of novel VSV-GP variants expressing immunomodulatory transgenes and can improve the assessment of anti-viral immunity in preclinical models.

Evaluating Antibody Pharmacokinetics as Prerequisite for Determining True Efficacy as Shown by Dual Targeting of PD-1 and CD96.

One important prerequisite for developing a therapeutic monoclonal antibody is to evaluate its in vivo efficacy. We tested the therapeutic potential of an anti-CD96 antibody alone or in combination with an anti-PD-1 antibody in a mouse colon cancer model. Early anti-PD-1 treatment significantly decreased tumor growth and the combination with anti-CD96 further increased the therapeutic benefit, while anti-CD96 treatment alone had no effect. In late therapeutic settings, the treatment combination resulted in enhanced CD8+ T cell infiltration of tumors and an increased CD8/Treg ratio. Measured anti-PD-1 concentrations were as expected in animals treated with anti-PD-1 alone, but lower at later time points in animals receiving combination treatment. Moreover, anti-CD96 concentrations dropped dramatically after 10 days and were undetectable thereafter in most animals due to the occurrence of anti-drug antibodies that were increasing antibody clearance. Comparison of the anti-PD-1 concentrations with tumor growth showed that higher antibody concentrations in plasma correlated with better therapeutic efficacy. The therapeutic effect of anti-CD96 treatment could not be evaluated, because plasma concentrations were too low. Our findings strongly support the notion of measuring both plasma concentration and anti-drug antibody formation throughout in vivo studies, in order to interpret pharmacodynamic data correctly.

A modular self-adjuvanting cancer vaccine combined with an oncolytic vaccine induces potent antitumor immunity.

Functional tumor-specific cytotoxic T cells elicited by therapeutic cancer vaccination in combination with oncolytic viruses offer opportunities to address resistance to checkpoint blockade therapy. Two cancer vaccines, the self-adjuvanting protein vaccine KISIMA, and the recombinant oncolytic vesicular stomatitis virus pseudotyped with LCMV-GP expressing tumor-associated antigens, termed VSV-GP-TAA, both show promise as a single agent. Here we find that, when given in a heterologous prime-boost regimen with an optimized schedule and route of administration, combining KISIMA and VSV-GP-TAA vaccinations induces better cancer immunity than individually. Using several mouse tumor models with varying degrees of susceptibility for viral replication, we find that priming with KISIMA-TAA followed by VSV-GP-TAA boost causes profound changes in the tumor microenvironment, and induces a large pool of poly-functional and persistent antigen-specific cytotoxic T cells in the periphery. Combining this heterologous vaccination with checkpoint blockade further improves therapeutic efficacy with long-term survival in the spectrum. Overall, heterologous vaccination with KISIMA and VSV-GP-TAA could sensitize non-inflamed tumors to checkpoint blockade therapy.