Determining mechanisms of resistance to αPD-1/PD-L1 immune-checkpoint immunotherapy is key to developing new treatment strategies. Cancer-associated fibroblasts (CAF) have many tumor-promoting ...functions and promote immune evasion through multiple mechanisms, but as yet, no CAF-specific inhibitors are clinically available. Here we generated CAF-rich murine tumor models (TC1, MC38, and 4T1) to investigate how CAFs influence the immune microenvironment and affect response to different immunotherapy modalities anticancer vaccination, TC1 (HPV E7 DNA vaccine), αPD-1, and MC38 and found that CAFs broadly suppressed response by specifically excluding CD8
T cells from tumors (not CD4
T cells or macrophages); CD8
T-cell exclusion was similarly present in CAF-rich human tumors. RNA sequencing of CD8
T cells from CAF-rich murine tumors and immunochemistry analysis of human tumors identified significant upregulation of CTLA-4 in the absence of other exhaustion markers; inhibiting CTLA-4 with a nondepleting antibody overcame the CD8
T-cell exclusion effect without affecting Tregs. We then examined the potential for CAF targeting, focusing on the ROS-producing enzyme NOX4, which is upregulated by CAF in many human cancers, and compared this with TGFβ1 inhibition, a key regulator of the CAF phenotype. siRNA knockdown or pharmacologic inhibition GKT137831 (Setanaxib) of NOX4 "normalized" CAF to a quiescent phenotype and promoted intratumoral CD8
T-cell infiltration, overcoming the exclusion effect; TGFβ1 inhibition could prevent, but not reverse, CAF differentiation. Finally, NOX4 inhibition restored immunotherapy response in CAF-rich tumors. These findings demonstrate that CAF-mediated immunotherapy resistance can be effectively overcome through NOX4 inhibition and could improve outcome in a broad range of cancers. SIGNIFICANCE: NOX4 is critical for maintaining the immune-suppressive CAF phenotype in tumors. Pharmacologic inhibition of NOX4 potentiates immunotherapy by overcoming CAF-mediated CD8
T-cell exclusion. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/9/1846/F1.large.jpg.
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DNA vaccination can elicit the production of anti-tumor antibodies, thus obviating the need to continuously administer passive antibody. This vaccination strategy is particularly important where ...antibodies have proven to be effective anti-tumor agents. To amplify antibody responses against weak tumor antigens, we previously designed DNA-fusion vaccines incorporating tumor sequences linked to microbial genes. By using a safe idiotypic (Id) antigen from a B cell tumor fused to a fragment C (FrC) sequence from tetanus toxin, we induced both anti-Id and anti-FrC antibodies. It was important to determine whether the antigen itself, either injected or released from residual tumor cells, would boost the antibody response. Id protein not only failed to boost the response, but permanently and rapidly inhibited it by ablating Id-specific memory B cells. In contrast, an Id protein-FrC conjugate boosted both Id-specific and FrC-specific responses. Strikingly, the depletion of CD4+T cells converted the Id protein-FrC conjugate vaccine into an inhibitor. These findings support the hypothesis that the activation of memory B cells by a DNA vaccine encoding a protein antigen, in the presence of the protein itself, depends completely on T cell help. Furthermore, by using knockout mice, we have shown that inhibition of the Id-specific memory B cells by the Id protein is largely independent of the FcγRIIB and, hence, independent of immune complexes. The principles revealed by using a DNA vaccine have implications for all cancer vaccines designed to induce and maintain antibody responses against weak autologous tumor antigens.
Myofibroblastic cancer-associated fibroblast (myoCAF)-rich tumors generally contain few T cells and respond poorly to immune-checkpoint blockade. Although myoCAFs are associated with poor outcome in ...most solid tumors, the molecular mechanisms regulating myoCAF accumulation remain unclear, limiting the potential for therapeutic intervention. Here, we identify ataxia-telangiectasia mutated (ATM) as a central regulator of the myoCAF phenotype. Differentiating myofibroblasts in vitro and myoCAFs cultured ex vivo display activated ATM signaling, and targeting ATM genetically or pharmacologically could suppress and reverse differentiation. ATM activation was regulated by the reactive oxygen species-producing enzyme NOX4, both through DNA damage and increased oxidative stress. Targeting fibroblast ATM in vivo suppressed myoCAF-rich tumor growth, promoted intratumoral CD8 T-cell infiltration, and potentiated the response to anti-PD-1 blockade and antitumor vaccination. This work identifies a novel pathway regulating myoCAF differentiation and provides a rationale for using ATM inhibitors to overcome CAF-mediated immunotherapy resistance.
ATM signaling supports the differentiation of myoCAFs to suppress T-cell infiltration and antitumor immunity, supporting the potential clinical use of ATM inhibitors in combination with checkpoint inhibition in myoCAF-rich, immune-cold tumors.
Vaccination with DNA that encodes cancer antigens is a simple and convenient way to raise immunity against cancer and has already shown promise in the clinical setting. Conventional plasmid DNA is ...commonly used which together with the encoded antigen also includes bacterial immunostimulatory CpG motifs to target the DNA sensor Toll-like receptor 9. Recently DNA vaccines using doggybone DNA (dbDNA™), have been developed without the use of bacteria. The cell-free process relies on the use of Phi29 DNA polymerase to amplify the template followed by protelomerase TelN to complete individual closed linear DNA. The resulting DNA contains the required antigenic sequence, a promoter and a poly A tail but lacks bacterial sequences such as an antibiotic resistance gene, prompting the question of immunogenicity. Here we compared the ability of doggybone DNA vaccine with plasmid DNA vaccine to induce adaptive immunity using clinically relevant oncotargets E6 and E7 from HPV. We demonstrate that despite the inability to trigger TLR9, doggybone DNA was able to induce similar levels of cellular and humoral immunity as plasmid DNA, with suppression of established TC-1 tumours.
Antigen-specific CD4⁺ T cells are central to natural and vaccine-induced immunity. An ongoing antigen-specific T-cell response can, however, influence surrounding T cells with unrelated antigen ...specificities. We previously observed this bystander effect in healthy human subjects following recall vaccination with tetanus toxoid (TT). Since this interplay could be important for maintenance of memory, we have moved to a mouse model for further analysis. We investigated whether boosting memory CD4⁺ T cells against TT in vivo would influence injected CD4⁺ TCR transgenic T cells (OT-II) specific for an unrelated OVA peptide. If OT-II cells were pre-activated with OVA peptide in vitro, these cells showed a bystander proliferative response during the ongoing parallel TT-specific response. Bystander proliferation was dependent on boosting of the TT-specific memory response in the recipients, with no effect in naive mice. Bystander stimulation was also proportional to the strength of the TT-specific memory T-cell response. T cells activated in vitro displayed functional receptors for IL-2 and IL-7, suggesting these as potential mediators. This crosstalk between a stimulated CD4⁺ memory T-cell response and CD4⁺ T cells activated by an unrelated antigen could be important in human subjects continually buffeted by environmental antigens.
Background
Human papilloma virus (HPV) infects keratinocytes of the skin and mucous membranes, and is associated with the induction of cutaneous warts and malignancy. Warts can induce significant ...morbidity and disability but most therapies, including cryotherapy, laser, and radiofrequency devices showlowefficacy and induce discomfort through tissue destruction. Microwaves are readily capable of passing through highly keratinised skin to deliver energy and induce heating of the tissue in a highly controllable, uniform manner.
Objectives
To determine the effects of microwave on cutaneous HPV infection.
Materials & methods
We undertook a pilot study of microwave therapy to the skin in 32 consecutive individuals with 52 recalcitrant long-lived viral cutaneous warts. Additionally, we undertook a molecular characterisation of the effects of microwaves on the skin.
Results
Tissue inflammation was minimal, but 75.9% of lesions cleared which compares favourably with previous studies showing a clearance rate of 23-33% for cryotherapy or salicylic acid. We show that microwaves specifically induce dendritic cell cross-presentation of HPV antigen to CD8+ T cells and suggest that IL-6 may be important for DC IRF1 and IRF4 modulation to enhance this process.
Conclusion
Keratinocyte-skin dendritic cell cross-talk is integral to host defence against HPV infections, and this pilot study supports the concept of microwave induction of anti-HPV immunity which offers a promising approach for treatment of HPV-induced viral warts and potentially HPV-related cancers.
Passive antibody therapy for cancer is an effective but costly treatment modality. Induction of therapeutically potent anticancer antibodies by active vaccination is an attractive alternative but has ...proven challenging in cancer due to tolerogenic pressure in patients. Here, we used the clinically relevant cancer target Her2, known to be susceptible to targeting by antibody therapy, to demonstrate how potent antibody can be induced by vaccination. A novel 44kD Her2 protein fragment was generated and found to be highly effective at inducing anti-Her2 antibody including trastuzumab-like reactivities. In the tolerant and spontaneous BALB-neuT mouse model of metastatic breast cancer this Her2-targeting vaccine was only effective if the fragment was conjugated to a foreign immunogenic carrier; Fragment C of tetanus toxin. Only the conjugate vaccine induced high affinity anti-Her2 antibody of multiple isotypes and suppressed tumor development. The magnitude of CD4
+
T-cell help and breadth of cytokines secreted by the CD4
+
T helper (Th) cells induced to the foreign antigen was critical. We used a highly efficient plant-based bio-manufacturing process for protein antigens, magnICON, for vaccine expression, to underpin feasibility of future clinical testing. Hence, our novel Her2-targeting conjugate vaccine combines preclinical efficacy with clinical deliverability, thus setting the scene for therapeutic testing.
The cancer/testis antigen NY‐ESO‐1 contains an immunodominant HLA‐A2‐binding peptide (SLLMWITQC), designated S9C, an attractive target for vaccination against several human cancers. As cysteine ...contains a reactive SH, the oxidation status of exogenous synthetic peptide is uncertain. We have designed tolerance‐breaking DNA fusion vaccines incorporating a domain of tetanus toxin fused to tumor‐derived peptide sequences (p.DOM‐peptide), placed at the C‐terminus for optimal immunogenicity. In a “humanized” HLA‐A2 preclinical model, p.DOM‐S9C primed S9C‐specific CD8+ T cells more effectively than adjuvanted synthetic peptide. A DNA vaccine encoding the full NY‐ESO‐1 sequence alone induced only weak S9C‐specific responses, amplified by addition of DOM sequence. The analog peptide (SLLMWITQL) also primed peptide‐specific CD8+ T cells, again increased by DNA delivery. Importantly, T cells induced by S9C‐encoding DNA vaccines killed tumor cells expressing endogenous NY‐ESO‐1. Only a fraction of T cells induced by the S9L‐encoding DNA vaccines was able to recognize S9C and kill tumor cells. These data indicate that DNA vaccines mimic posttranslational modifications of SH‐containing peptides expressed by tumor cells. Instability of synthetic peptides and the potential dangers of analog peptides contrast with the ability of DNA vaccines to induce high levels of tumor‐lytic peptide‐specific CD8+ T cells. These findings encourage clinical exploration of this vaccine strategy to target NY‐ESO‐1.
What's new?
Vaccine‐targeted cancer peptides containing amino acids such as cysteine, can be post‐translationally modified in unpredictable ways by tumor cells, making peptide design difficult. In contrast, DNA fusion gene vaccines deliver peptide in a form which mimics tumor cell processing and induce high levels of tumor‐lytic CD8+ T cells. This simple strategy obviates the need for analogue peptides, which, even when optimized, generate both relevant and irrelevant responses.
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