Faculty Bibliography

Through the action of two virus-encoded decapping enzymes (D9 and D10) that remove protective caps from mRNA 5'-termini, Vaccinia virus (VACV) accelerates mRNA decay and limits activation of host defenses. D9- or D10-deficient VACV are markedly attenuated in mice and fail to counter cellular double-stranded RNA-responsive innate immune effectors, including PKR. Here, we capitalize upon this phenotype and demonstrate that VACV deficient in either decapping enzyme are effective oncolytic viruses. Significantly, D9- or D10-deficient VACV displayed anti-tumor activity against syngeneic mouse tumors of different genetic backgrounds and human hepatocellular carcinoma xenografts. Furthermore, D9- and D10-deficient VACV hyperactivated the host anti-viral enzyme PKR in non-tumorigenic cells compared to wild-type virus. This establishes a new genetic platform for oncolytic VACV development that is deficient for a major pathogenesis determinant while retaining viral genes that support robust productive replication like those required for nucleotide metabolism. It further demonstrates how VACV mutants unable to execute a fundamental step in virus-induced mRNA decay can be unexpectedly translated into a powerful anti-tumor therapy.

Background: Although much clinical progress has been made in harnessing the immune system to recognize and target cancer, there is still a significant lack of an understanding of how tumors evade immune recognition and the mechanisms that drive tumor resistance to both T cell and checkpoint blockade immunotherapy. Our objective is to understand how tumor-mediated signaling through inhibitory receptors, including PD-1, combine to affect the process of T cell recognition of tumor antigen and activation signaling, with the goal of understanding the basis of resistance to PD-1 blockade and the potential identification of new molecular targets to enable T cells to overcome dysfunction mediated by multiple inhibitory receptors.
Methods and Results: We show that Lck activity affects T cell sensitivity and influences the probability of inducing effector function [1]. Under non-activating conditions, Lck and Shp-1 phosphorylation and activity vary based on CD8+ memory T cell phenotype. Shp-1 interaction with Lck under non-activation conditions can also vary, as suggested by our results showing decreased Shp-1 S591 phosphorylation, which affects Shp-1 localization and correlates with increased Shp-1 colocalization with Lck. Further, we showed that Shp-1 directly influences Lck activity under non-activating conditions, as inhibition of Shp-1 leads to increased Lck activity. Importantly, inhibition of Shp- 1/2, a major mediator of PD-1 signaling, targeting CD28 and Lck [2], prior to activation leads to increased T cell cytotoxic effector function. Our proteomics-based analysis of patient T cells identified both mediators of PD-1 signaling and signaling components and pathways associated with blockade resistance. It has generally been thought that TCR and CD8 binding depend mainly on their ectodomain interactions with pMHC. We have shown, however, that Lck-CD8 binding [3] and Lck activity [4] are required for upregulated CD8 binding to prebound TCR-pMHC complex. Therefore, the cytoplasmic associations of Lck with CD8 and Zap-70, as well as CD3 with Zap-70 may influence formation and stability of the TCRpMHCCD8 complex. To determine the mechanistic basis of PD-1 inhibition of TCR-pMHCCD8 binding we utilized 2D affinity combined with Biomembrane Force Probe (BFP) measurements[5, 6] and showed that PD-1 directly suppresses TCR pMHCCD8 binding. Our data also revealed that TCR-pMHC binding was independent of PD-1-PD-L1, but TCRpMHCCD8 binding was suppressed by PD-1-PD-L1 binding demonstrating negative cooperativity, as fewer bonds formed than the sum of bonds formed by each interaction alone.
Conclusion(s): Together, our results show that the activities of TCRproximal signaling components affect T cell mechanosensing and sensitivity at the earliest stages of antigen recognition and are influenced by PD-1. Targeting these interactions may enhance tumor-specific T cell sensitivity for cancer immunotherapy and understanding the basis of resistance to PD-1 blockade to potentially allow identification of new molecular targets to enable T cells to overcome dysfunction mediated by multiple inhibitory receptors

Cancers are infiltrated with antitumor CD8+ T cells that arise during tumor growth, but are defective in effector phase functions because of the suppressive microenvironment. The reactivation of TILs can result in tumor destruction, showing that lytic dysfunction in CD8+ tumor-infiltrating lymphocytes (TILs) permits tumor growth. Like all memory T cells, TILs express inhibitory signaling receptors (aka checkpoint inhibitor molecules) that downregulate TCR-mediated signal transduction upon TIL interaction with cells expressing cognate ligands, thereby restricting cell activation and preventing the effector phase. Previously we identified a novel murine CD8+ TIL inhibitory signaling receptor, protocadherin-18, and showed that it interacts with p56lck kinase to abrogate proximal TCR signaling. Here we show that TILs from mice deleted in protocadherin-18 had enhanced antitumor activity and that co-blockade of PD-1 and protocadherin-18 in wild-type mice significantly enhanced TIL effector phase function. These results define an important role for protocadherin-18 in antitumor T-cell activity.

CD8+ T cells develop increased sensitivity following Ag experience, and differences in sensitivity exist between T cell memory subsets. How differential TCR signaling between memory subsets contributes to sensitivity differences is unclear. We show in mouse effector memory T cells (TEM) that >50% of lymphocyte-specific protein tyrosine kinase (Lck) exists in a constitutively active conformation, compared with <20% in central memory T cells (TCM). Immediately proximal to Lck signaling, we observed enhanced Zap-70 phosphorylation in TEM following TCR ligation compared with TCM Furthermore, we observed superior cytotoxic effector function in TEM compared with TCM, and we provide evidence that this results from a lower probability of TCM reaching threshold signaling owing to the decreased magnitude of TCR-proximal signaling. We provide evidence that the differences in Lck constitutive activity between CD8+ TCM and TEM are due to differential regulation by SH2 domain-containing phosphatase-1 (Shp-1) and C-terminal Src kinase, and we use modeling of early TCR signaling to reveal the significance of these differences. We show that inhibition of Shp-1 results in increased constitutive Lck activity in TCM to levels similar to TEM, as well as increased cytotoxic effector function in TCM Collectively, this work demonstrates a role for constitutive Lck activity in controlling Ag sensitivity, and it suggests that differential activities of TCR-proximal signaling components may contribute to establishing the divergent effector properties of TCM and TEM. This work also identifies Shp-1 as a potential target to improve the cytotoxic effector functions of TCM for adoptive cell therapy applications.

Myeloid-derived suppressor cells (MDSCs) have emerged as major regulators of immune responses in cancer and other pathological conditions. In recent years, ample evidence supports key contributions of MDSC to tumour progression through both immune-mediated mechanisms and those not directly associated with immune suppression. MDSC are the subject of intensive research with >500 papers published in 2015 alone. However, the phenotypic, morphological and functional heterogeneity of these cells generates confusion in investigation and analysis of their roles in inflammatory responses. The purpose of this communication is to suggest characterization standards in the burgeoning field of MDSC research.

Although counteracting innate defenses allows oncolytic viruses (OVs) to better replicate and spread within tumors, CD8(+) T-cells restrict their capacity to trigger systemic anti-tumor immune responses. Herpes simplex virus-1 (HSV-1) evades CD8(+) T-cells by producing ICP47, which limits immune recognition of infected cells by inhibiting the transporter associated with antigen processing (TAP). Surprisingly, removing ICP47 was assumed to benefit OV immuno-therapy, but the impact of inhibiting TAP remains unknown because human HSV-1 ICP47 is not effective in rodents. Here, we engineer an HSV-1 OV to produce bovine herpesvirus UL49.5, which unlike ICP47, antagonizes rodent and human TAP. Significantly, UL49.5-expressing OVs showed superior efficacy treating bladder and breast cancer in murine models that was dependent upon CD8(+) T-cells. Besides injected subcutaneous tumors, UL49.5-OV reduced untreated, contralateral tumor size and metastases. These findings establish TAP inhibitor-armed OVs that evade CD8(+) T-cells as an immunotherapy strategy to elicit potent local and systemic anti-tumor responses.

Type 1 diabetes is an autoimmune disease in which insulin-producing pancreatic islet beta cells are the target of self-reactive B and T cells. T cells reactive with epitopes derived from insulin and/or IGRP are critical for the initiation and maintenance of disease, but T cells reactive with other islet antigens likely have an essential role in disease progression. We sought to identify candidate CD8(+) T cell epitopes that are pathogenic in type 1 diabetes. Proteins that elicit autoantibodies in human type 1 diabetes were analyzed by predictive algorithms for candidate epitopes. Using several different tolerizing regimes using synthetic peptides, two new predicted tolerogenic CD8(+) T cell epitopes were identified in the murine homolog of the major human islet autoantigen zinc transporter ZnT8 (aa 158-166 and 282-290) and one in a non-beta cell protein, dopamine beta-hydroxylase (aa 233-241). Tolerizing vaccination of NOD mice with a cDNA plasmid expressing full-length proinsulin prevented diabetes, whereas plasmids encoding ZnT8 and DbetaH did not. However, tolerizing vaccination of NOD mice with the proinsulin plasmid in combination with plasmids expressing ZnT8 and DbetaH decreased insulitis and enhanced prevention of disease compared to vaccination with the plasmid encoding proinsulin alone.