The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates cellular processes in cancer and immunity, including innate immune cell development and effector function. However, the transcriptional repertoire through which AHR mediates these effects remains largely unexplored. To elucidate the transcriptional elements directly regulated by AHR in natural killer (NK) cells, we performed RNA and chromatin immunoprecipitation sequencing on NK cells exposed to AHR agonist or antagonist. We show that mature peripheral blood NK cells lack AHR, but its expression is induced by Stat3 during interleukin-21–driven activation and proliferation, coincident with increased NCAM1 (CD56) expression resulting in a CD56bright phenotype. Compared with control conditions, NK cells expanded in the presence of the AHR antagonist, StemRegenin-1, were unaffected in proliferation or cytotoxicity, had no increase in NCAM1 transcription, and maintained the CD56dim phenotype. However, it showed altered expression of 1004 genes including those strongly associated with signaling pathways. In contrast, NK cells expanded in the presence of the AHR agonist, kynurenine, showed decreased cytotoxicity and altered expression of 97 genes including those strongly associated with oxidative stress and cellular metabolism. By overlaying these differentially expressed genes with AHR chromatin binding, we identified 160 genes directly regulated by AHR, including hallmark AHR targets AHRR and CYP1B1 and known regulators of phenotype, development, metabolism, and function such as NCAM1, KIT, NQO1, and TXN. In summary, we define the AHR transcriptome in NK cells, propose a model of AHR and Stat3 coregulation, and identify potential pathways that may be targeted to overcome AHR-mediated immune suppression.

Background T cell checkpoint receptors are expressed when T cells are activated, and modulation of the expression or signaling of these receptors can alter the function of T cells and their antitumor efficacy. We previously found that T cells activated with cognate antigen had increases in the expression of PD-1, and this was attenuated in the presence of multiple toll-like receptor (TLR) agonists, notably TLR3 plus TLR9. In the current report, we sought to investigate whether combining TLR agonists with immune checkpoint blockade can further augment vaccine-mediated T cell antitumor immunity in murine tumor models. Methods TLR agonists (TLR3 plus TLR9) and immune checkpoint inhibitors (antibodies targeting PD-1, CTLA-4, LAG-3, TIM-3 or VISTA) were combined and delivered with vaccines or vaccine-activated CD8+T cells to E.G7-OVA or MyC-CaP tumor-bearing mice. Tumors were assessed for growth and then collected and analyzed by flow cytometry. Results Immunization of E.G7-OVA tumor-bearing mice with SIINFEKL peptide vaccine, coadministered with TLR agonists and αCTLA-4, demonstrated greater antitumor efficacy than immunization with TLR agonists or αCTLA-4 alone. Conversely, the antitumor efficacy was abrogated when vaccine and TLR agonists were combined with αPD-1. TLR agonists suppressed PD-1 expression on regulatory T cells (Tregs) and activated this population. Depletion of Tregs in tumor-bearing mice led to greater antitumor efficacy of this combination therapy, even in the presence of αPD-1. Combining vaccination with TLR agonists and αCTLA-4 or αLAG-3 showed greater antitumor than with combinations with αTIM-3 or αVISTA. Conclusion The combination of TLR agonists and αCTLA-4 or αLAG-3 can further improve the efficacy of a cancer vaccine, an effect not observed using αPD-1 due to activation of Tregs when αPD-1 was combined with TLR3 and TLR9 agonists. These data suggest that optimal combinations of TLR agonists and immune checkpoint blockade may improve the efficacy of human anticancer vaccines.

Metastatic castration-resistant prostate cancer (mCRPC) is a challenging disease to treat, with poor outcomes for patients. One antitumor vaccine, sipuleucel-T, has been approved as a treatment for mCRPC. DNA vaccines are another form of immunotherapy under investigation. DNA immunizations elicit antigen-specific T cells that cause tumor cell lysis, which should translate to meaningful clinical responses. They are easily amenable to design alterations, scalable for large-scale manufacturing, and thermo-stable for easy transport and distribution. Hence, they offer advantages over other vaccine formulations. However, clinical trials with DNA vaccines as a monotherapy have shown only modest clinical effects against tumors. Standard therapies for CRPC including androgen-targeted therapies, radiation therapy and chemotherapy all have immunomodulatory effects, which combined with immunotherapies such as DNA vaccines, could potentially improve treatment. In addition, many investigational drugs are being developed which can augment antitumor immunity, and together with DNA vaccines can further enhance antitumor responses in preclinical models. We reviewed the literature available prior to July 2020 exploring the use of DNA vaccines in the treatment of prostate cancer. We also examined various approved and experimental therapies that could be combined with DNA vaccines to potentially improve their antitumor efficacy as treatments for mCRPC.

Background: We have previously shown that immune checkpoint receptors, including PD-1, are upregulated on T cells at the time of their activation, and that blockade of these receptors can improve the efficacy of antitumor vaccines. In the present study, we sought to determine whether, and by what mechanisms, the timing of PD-1 blockade with respect to vaccination affects antitumor T cell function. Methods: TRAMP-C1 or E.G7-OVA tumor-bearing mice received PD-1 blockade at different timing intervals with a tumor-associated antigen vaccine. Tumor growth, survival, and immune-infiltrating populations were assessed. In vitro models of T cell activation using OT-I T cells and PD-(L)1 axis disruption with a PD-1 blocking antibody or PD-L1 ^KO dendritic cells were used. Results: Mice receiving PD-1 blockade at the time of T cell activation with vaccine had better antitumor outcomes in comparison to mice receiving PD-1 blockade before or after immunization. T cells activated in vitro in the presence of PD-(L)1 axis disruption had a more differentiated, functional phenotype with decreased CD28 and CCR7 expression and increased production of the Tc1 cytokines IL-2, TNFα, and IFNγ. Intriguingly, a small subset of undifferentiated cells (CD28+) was of a stem-like Tc17 phenotype (IL-17α+, TCF1+). Tumor-bearing mice receiving T cells activated in the presence of PD-(L)1-axis disruption had better antitumor outcomes and a greater number of complete responses. Conclusions: These data indicate that PD-1 blockade, when used with antitumor vaccines, acts primarily at the time of T cell activation, not exclusively within the tumor microenvironment. Consequently, PD-1 blockade may be best used when delivered concurrently with T cell activating agents such as vaccines.

Immunotherapy is now an established therapy for nearly a third of patients with cancer. Most therapies, typically using cytokines or checkpoint blockade therapy, rely on global activation of immune effector cells. The ability of vaccines to activate specific populations of cells has led to a renewed interest in their ability to treat cancers, either alone or with other immune therapies or other conventional therapies. The COVID-19 pandemic sparked a new interest in nucleic acid vaccines with the development of new technologies and the short manufacturing time for vaccine implementation. Nucleic acid-based cancer vaccines have been studied for decades, but have shown modest anti-tumor efficacy as monotherapies, as many of these vaccines encode for shared tumor-associated antigens (TAAs) and must overcome immune tolerance. New developments, technologies, routes of delivery, and combination therapies have paved the way for new approaches and clinical trials involving nucleic acid vaccines for the treatment of cancer. Here we review mRNA and pDNA vaccines for the treatment of cancer, including similarities and differences in their mechanisms of action, an overview of these treatment modalities in preclinical and clinical studies, methods to improve these vaccine strategies, and exciting new combination approaches in development.

Peptide vaccines offer a direct way to initiate an immunogenic response to a defined antigen epitope. However, peptide vaccines are unstable in vivo, subject to rapid enzymatic proteolysis. Replacement of an α-amino acid residue with a homologous β-amino acid residue (native side chain, but backbone extended by a single CH₂ unit) impairs proteolysis at nearby amide bonds. Therefore, antigen analogues containing α-to-β replacements have been examined for functional mimicry of native all-α antigens. Another group previously took this approach in the ovalbumin (OVA) antigen model by evaluating single α-to-β analogues of the murine major histocompatibility complex (MHC) I-restricted peptide SIINFEKL. We re-examined this set of α/β SIINFEKL antigens. We tested the susceptibility to proteolysis in mouse serum and their ability to activate OVA-antigen-specific CD8 T cells in vitro. Additionally, we tested the α/β antigens in vivo for their ability to induce an antigen-specific immunogenic response in naïve mice and in OVA-expressing tumor-bearing mice. The α/β antigens were comparable to the native antigen in their susceptibility to proteolysis in serum. Each α/β antigen was capable of activating antigen-specific CD8 T cells in vitro. However, antigen-specific CD8 T cells induced against α/β antigens in vivo were not cross-reactive to the native antigen. Moreover, immunization with α/β analogues did not elicit anti-tumor effects in tumor-bearing mice. We conclude that even though α/β analogues of the SIINFEKL antigen can elicit a T cell-based response, this class of backbone-modified peptides is not promising from the perspective of antitumor vaccine development.