PD-1-targeted therapies have shown modest antiviral effects in preclinical models of chronic viral infection. Thus, novel therapy protocols are necessary to enhance T cell immunity and viral control to overcome T cell dysfunction and immunosuppression. Here, we demonstrate that nanoparticle-based therapeutic vaccination improved PD-1-targeted therapy during chronic infection with Friend retrovirus (FV). Prevention of inhibitory signals by blocking PD-L1 in combination with therapeutic vaccination with nanoparticles containing the microbial compound CpG and a CD8+ T cell Gag epitope peptide synergistically enhanced functional virus-specific CD8+ T cell responses and improved viral clearance. We characterized the CD8+ T cell populations that were affected by this combination therapy, demonstrating that new effector cells were generated and that exhausted CD8+ T cells were reactivated at the same time. While CD8+ T cells with high PD-1 (PD-1hi) expression turned into a large population of granzyme B-expressing CD8+ T cells after combination therapy, CXCR5-expressing follicular cytotoxic CD8+ T cells also expanded to a high degree. Thus, our study describes a very efficient approach to enhance virus control and may help us to understand the mechanisms of combination immunotherapy reactivating CD8+ T cell immunity. A better understanding of CD8+ T cell immunity during combination therapy will be important for developing efficient checkpoint therapies against chronic viral infections and cancer.IMPORTANCE Despite significant efforts, vaccines are not yet available for every infectious pathogen, and the search for a protective approach to prevent the establishment of chronic infections, i.e., with HIV, continues. Immune checkpoint therapies targeting inhibitory receptors, such as PD-1, have shown impressive results against solid tumors. However, immune checkpoint therapies have not yet been licensed to treat chronic viral infections, since a blockade of inhibitory receptors alone provides only limited benefit, as demonstrated in preclinical models of chronic viral infection. Thus, there is a high interest in the development of potent combination immunotherapies. Here, we tested whether the combination of a PD-L1 blockade and therapeutic vaccination with functionalized nanoparticles is a potent therapy during chronic Friend retrovirus infection. We demonstrate that the combination therapy induced a synergistic reinvigoration of the exhausted virus-specific CD8+ T cell immunity. Taken together, our results provide further information on how to improve PD-1-targeted therapies during chronic viral infection and cancer.
Copyright © 2021 Knuschke et al.

Cutaneous squamous cell carcinoma (cSCC) development has been linked to immune dysfunctions but the mechanisms are still unclear. Here, we report a progressive infiltration of tumor-associated neutrophils (TANs) in precancerous and established cSCC lesions from chemically induced skin carcinogenesis. Comparative in-depth gene expression analyses identified a predominant protumor gene expression signature of TANs in lesions compared to their respective surrounding skin. In addition, in vivo depletion of neutrophils delayed tumor growth and significantly increased the frequency of proliferating IFN-γ (interferon-γ)-producing CD8+ T cells. Mechanisms that limited antitumor responses involved high arginase activity, production of reactive oxygen species (ROS) and nitrite (NO), and the expression of programmed death-ligand 1 (PD-L1) on TAN, concomitantly with an induction of PD-1 on CD8+ T cells, which correlated with tumor size. Our data highlight the relevance of targeting neutrophils and PD-L1-PD-1 (programmed death-1) interaction in the treatment of cSCC.

Synthesis of extracellular adenosine by the ectonucleotidases CD39 and CD73 represents an important pathway of immune suppression in the tumor microenvironment. Using two mouse models (RET transgenic melanoma and Panc02 orthotopic pancreatic adenocarcinoma), we identified an elevated frequency of ectonucleotidase-expressing T cells in tumors and spleens. Importantly, these ectonucleotidase-positive T cells also showed a pronounced expression of PD-1. Conversely, the PD-1+ T cell subsets in tumors contained substantially larger proportions of ectonucleotidase-expressing cells compared to their counterparts lacking PD-1 expression. Our in vitro experiments showed that the activation of normal T cells resulted in an increase in the CD39 expression. CD39+ and CD73+ T cells displayed effector or memory phenotypes and produced IFN-γ, thereby linking ectonucleotidase expression to T cell effector functions. An accumulation of conventional and regulatory T cells expressing CD39 and/or CD73 was also detected in the peripheral blood of patients with melanoma and pancreatic cancer. Moreover, we demonstrated a significant association between low frequencies of circulating CD73+CD8+ T cells and CD73+CD4+ regulatory T cells and better overall survival of melanoma patients. Tumor-derived soluble factors (in particular, TGF-β) significantly enhanced the frequencies of ectonucleotidase-expressing cells in mice. Our findings suggest that the upregulation of ectonucleotidase expression in T cells promotes extracellular adenosine accumulation and represents an important mechanism of homeostatic immune auto-regulation, which could be hijacked by tumors to evade anti-cancer immunity. Targeting CD39 and CD73 can open new avenues for cancer immunotherapy.
© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.

We have previously shown that an AAV6-based vaccine generates high levels of antigen-specific CD8+ T cells. Further modifications described here led to significantly increased levels of antigen-specific CD8+ and CD4+ T cells, enhanced formation of memory cells, and superior antigen-specific killing capacity in a murine model. By tracking reporter-gene-positive dendritic cells, we showed that they were directly targeted with modified AAV6 in vivo. Our vaccine's anti-cancer potential was evaluated with the antigen ovalbumin against a B16F10 melanoma cell line stably expressing ovalbumin. The vaccination showed superior protection in a murine model of metastatic melanoma. The vaccination significantly delayed solid tumor growth but did not completely prevent tumor development. We show that tumors in immunized mice escaped vaccine-induced killing by losing ovalbumin expression. The vaccine induced massive tumor infiltration with NK and CD8+ T cells with upregulated PD-1 expression. Thus, a vaccination of a combination of anti-PD-1 antibodies demonstrated significant improvement in the treatment efficacy. To summarize, we showed that a bioengineered AAV6-based vaccine elicits strong and long-lasting cellular and humoral responses against an encoded antigen. To increase AAV vaccine efficiency and mitigate tumor escape through antigen loss, we intended to target several antigens in combination with treatments targeting the tumor microenvironment.
© 2019 The Author(s).

RNA replicons are a promising platform technology for vaccines. To evaluate the potential of lipid nanoparticle-formulated replicons for delivery of HIV immunogens, we designed and tested an alphavirus replicon expressing a self-assembling protein nanoparticle immunogen, the glycoprotein 120 (gp120) germline-targeting engineered outer domain (eOD-GT8) 60-mer. The eOD-GT8 immunogen is a germline-targeting antigen designed to prime human B cells capable of evolving toward VRC01-class broadly neutralizing antibodies. Replicon RNA was encapsulated with high efficiency in 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-based lipid nanoparticles, which provided effective delivery in the muscle and expression of luciferase lasting ∼30 days in normal mice, contrasting with very brief and low levels of expression obtained by delivery of equivalent modified mRNA (modRNA). eOD-GT8 60-mer-encoding replicons elicited high titers of gp120-specific antibodies following a single injection in mice, and increased levels of antigen-specific germinal center B cells compared with protein immunization. Immunization of transgenic mice expressing human inferred-germline VRC01 heavy chain B cell receptors that are the targets of the eOD antigen led to priming of B cells and somatic hypermutation consistent with VRC01-class antibody development. Altogether, these data suggest replicon delivery of Env immunogens may be a promising avenue for HIV vaccine development.
Copyright © 2019 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.