FOXO3, estrogen receptor alpha, and androgen receptor impact tumor growth rate and infiltration of dendritic cell subsets differentially between male and female mice.

Tumors evade immune recognition and destruction in many ways including the creation of an immune-suppressive tumor microenvironment (TME). Dendritic cells (DC) that infiltrate the TME are tolerogenic, and suppress effector T cells and anti-tumor activity. Previous reports demonstrated that a key regulator of tolerance in DC is the transcription factor FOXO3. Gender disparity has been studied in cancer in relation to incidence, aggressiveness, and prognosis. Few studies have touched on the importance in relation to impact on the immune system. In the current study, we show that there are significant differences in tumor growth between males and females. Additionally, frequencies and the function of FOXO3 expressed by DC subsets that infiltrate tumors vary between genders. Our results show for the first time that DC FOXO3 expression and function is altered in females. In vitro results indicate that these differences may be the result of exposure to estrogen. These differences may be critical considerations for the enhancement of immunotherapy for cancer.

FOXO3-NF-κB RelA Protein Complexes Reduce Proinflammatory Cell Signaling and Function.

Tumor-associated myeloid cells, including dendritic cells (DCs) and macrophages, are immune suppressive. This study demonstrates a novel mechanism involving FOXO3 and NF-κB RelA that controls myeloid cell signaling and impacts their immune-suppressive nature. We find that FOXO3 binds NF-κB RelA in the cytosol, impacting both proteins by preventing FOXO3 degradation and preventing NF-κB RelA nuclear translocation. The location of protein-protein interaction was determined to be near the FOXO3 transactivation domain. In turn, NF-κB RelA activation was restored upon deletion of the same sequence in FOXO3 containing the DNA binding domain. We have identified for the first time, to our knowledge, a direct protein-protein interaction between FOXO3 and NF-κB RelA in tumor-associated DCs. These detailed biochemical interactions provide the foundation for future studies to use the FOXO3-NF-κB RelA interaction as a target to enhance tumor-associated DC function to support or enhance antitumor immunity.

Establishment of a murine hepatocellular carcinoma model by hydrodynamic injection and characterization of the immune tumor microenvironment.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignant neoplasms. Current treatments for HCC, such as tyrosine kinase inhibitors, have limited efficacy, highlighting the urgent need for better therapies. Immunotherapies, including anti-programmed death receptor 1 (PD-1) and anti-Cytotoxic T-lymphocyte associated protein 4 (CTLA-4), and more recently, the combination of anti-PD-L1 and anti-vascular endothelial growth factor (VEGF) monoclonal antibodies, have shown efficacy against HCC, resulting in Food and Drug Administration (FDA) approval. However, these immunotherapies only show efficiency in a small proportion of patients, meaning there is a great need to improve and optimize treatments against HCC. Accurate animal models that mimic human HCC are necessary to help better understand the nature of these tumors, which in turn will allow the development and testing of new treatments. Existing pre-clinical HCC models can be divided into non-genetic and genetic models. Non-genetic models involve implanting human or murine HCC cell lines or inducing tumors using chemical compounds or dietary modifications. These models have limitations, including slow tumor development and a lack of resemblance to human HCC. Genetic models, on the other hand, manipulate gene expression to induce HCC in mice and provide a better understanding of the effects of specific genes on tumor development. One method commonly used to generate HCC is hydrodynamic tail vein injection (HTVI), which consists of the delivery of oncogenes directly to the liver, resulting in expression and subsequent hepatocyte transformation. Usually, Sleeping Beauty transposase-containing plasmids are used to achieve stable and long-term gene expression. Once the HCC tumor is generated, and a proper tumor microenvironment (TME) is established, it is important to study the immune compartment of the TME, which plays a crucial role in HCC development and response to treatment. Techniques like flow cytometry can be used to analyze the immune cell populations in HCC tumors and assess their impact on tumor development and survival in mice. In this article, we thoroughly describe an example of the methodology to successfully generate HCC murine models via HTVI, and we propose a way to characterize the immune TME by flow cytometry.

Tethered IL15-IL15Rα augments antitumor activity of CD19 CAR-T cells but displays long-term toxicity in an immunocompetent lymphoma mouse model.

BACKGROUND: Adoptive cell therapy using genetically modified T cells to express chimeric antigen receptors (CAR-T) has shown encouraging results, particularly in certain blood cancers. Nevertheless, over 40% of B cell malignancy patients experience a relapse after CAR-T therapy, likely due to inadequate persistence of the modified T cells in the body. IL15, known for its pro-survival and proliferative properties, has been suggested for incorporation into the fourth generation of CAR-T cells to enhance their persistence. However, the potential systemic toxicity associated with this cytokine warrants further evaluation. METHODS: We analyzed the persistence, antitumor efficacy and potential toxicity of anti-mouse CD19 CAR-T cells which express a membrane-bound IL15-IL15Rα chimeric protein (CD19/mbIL15q CAR-T), in BALB/c mice challenged with A20 tumor cells as well as in NSG mice. RESULTS: Conventional CD19 CAR-T cells showed low persistence and poor efficacy in BALB/c mice treated with mild lymphodepletion regimens (total body irradiation (TBI) of 1 Gy). CD19/mbIL15q CAR-T exhibits prolonged persistence and enhanced in vivo efficacy, effectively eliminating established A20 B cell lymphoma. However, this CD19/mbIL15q CAR-T displays important long-term toxicities, with marked splenomegaly, weight loss, transaminase elevations, and significant inflammatory findings in some tissues. Mice survival is highly compromised after CD19/mbIL15q CAR-T cell transfer, particularly if a high TBI regimen is applied before CAR-T cell transfer. CONCLUSION: Tethered IL15-IL15Rα augments the antitumor activity of CD19 CAR-T cells but displays long-term toxicity in immunocompetent mice. Inducible systems to regulate IL15-IL15Rα expression could be considered to control this toxicity.

Improvement of cognitive function in wild-type and Alzheimer´s disease mouse models by the immunomodulatory properties of menthol inhalation or by depletion of T regulatory cells.

A complex network of interactions exists between the olfactory, immune and central nervous systems. In this work we intend to investigate this connection through the use of an immunostimulatory odorant like menthol, analyzing its impact on the immune system and the cognitive capacity in healthy and Alzheimer's Disease Mouse Models. We first found that repeated short exposures to menthol odor enhanced the immune response against ovalbumin immunization. Menthol inhalation also improved the cognitive capacity of immunocompetent mice but not in immunodeficient NSG mice, which exhibited very poor fear-conditioning. This improvement was associated with a downregulation of IL-1ß and IL-6 mRNA in the brain´s prefrontal cortex, and it was impaired by anosmia induction with methimazole. Exposure to menthol for 6 months (1 week per month) prevented the cognitive impairment observed in the APP/PS1 mouse model of Alzheimer. Besides, this improvement was also observed by the depletion or inhibition of T regulatory cells. Treg depletion also improved the cognitive capacity of the APPNL-G-F/NL-G-F Alzheimer´s mouse model. In all cases, the improvement in learning capacity was associated with a downregulation of IL-1ß mRNA. Blockade of the IL-1 receptor with anakinra resulted in a significant increase in cognitive capacity in healthy mice as well as in the APP/PS1 model of Alzheimer´s disease. These data suggest an association between the immunomodulatory capacity of smells and their impact on the cognitive functions of the animals, highlighting the potential of odors and immune modulators as therapeutic agents for CNS-related diseases.

The role of dendritic cells in the immune niche of the peritoneum.

Dendritic cells (DCs) are professional antigen presenting cells that play an important role in the induction of T cell responses. Different subsets (cDC1s, cDC2s, pDCs, and moDCs) were described based on the expression of different surface markers and functions. In the context of peritoneum, DCs are also a key population cell orchestrating immune responses against pathogens, malignant cells and tissue-damage. Furthermore, they play an important role in the promotion of an anti-inflammatory microenvironment, which is necessary to maintain tolerance and adipocyte homeostasis. The aim of this review is to summarize the current knowledge of the functional and phenotypic features of peritoneal DCs and shed some light on the importance of these cells within this unique cavity and its associated components: the omentum, the mesentery and gut-associated lymphoid tissue (GALT).

Impact of tumor microenvironment on adoptive T cell transfer activity.

Recent advances in immunotherapy have revolutionized the treatment of cancer. The use of adoptive cell therapies (ACT) such as those based on tumor infiltrating lymphocytes (TILs) or genetically modified cells (transgenic TCR lymphocytes or CAR-T cells), has shown impressive results in the treatment of several types of cancers. However, cancer cells can exploit mechanisms to escape from immunosurveillance resulting in many patients not responding to these therapies or respond only transiently. The failure of immunotherapy to achieve long-term tumor control is multifactorial. On the one hand, only a limited percentage of the transferred lymphocytes is capable of circulating through the bloodstream, interacting and crossing the tumor endothelium to infiltrate the tumor. Metabolic competition, excessive glucose consumption, the high level of lactic acid secretion and the extracellular pH acidification, the shortage of essential amino acids, the hypoxic conditions or the accumulation of fatty acids in the tumor microenvironment (TME), greatly hinder the anti-tumor activity of the immune cells in ACT therapy strategies. Therefore, there is a new trend in immunotherapy research that seeks to unravel the fundamental biology that underpins the response to therapy and identifies new approaches to better amplify the efficacy of immunotherapies. In this review we address important aspects that may significantly affect the efficacy of ACT, indicating also the therapeutic alternatives that are currently being implemented to overcome these drawbacks.

Overcoming T cell dysfunction in acidic pH to enhance adoptive T cell transfer immunotherapy.

The high metabolic activity and insufficient perfusion of tumors leads to the acidification of the tumor microenvironment (TME) that may inhibit the antitumor T cell activity. We found that pharmacological inhibition of the acid loader chloride/bicarbonate anion exchanger 2 (Ae2), with 4,4'-diisothiocyanatostilbene-2,2'-disulfonicacid (DIDS) enhancedCD4+ andCD8+ T cell function upon TCR activation in vitro, especially under low pH conditions. In vivo, DIDS administration delayed B16OVA tumor growth in immunocompetent mice as monotherapy or when combined with adoptive T cell transfer of OVA-specificT cells. Notably, genetic Ae2 silencing in OVA-specificT cells improvedCD4+/CD8+ T cell function in vitro as well as their antitumor activity in vivo. Similarly, genetic modification of OVA-specificT cells to overexpress Hvcn1, a selectiveH+ outward current mediator that prevents cell acidification, significantly improved T cell function in vitro, even at low pH conditions. The adoptive transfer of OVA-specificT cells overexpressing Hvcn1 exerted a better antitumor activity in B16OVA tumor-bearingmice. Hvcn1 overexpression also improved the antitumor activity of CAR T cells specific for Glypican 3 (GPC3) in mice bearing PM299L-GPC3tumors. Our results suggest that preventing intracellular acidification by regulating the expression of acidifier ion channels such as Ae2 or alkalinizer channels like Hvcn1 in tumor-specificlymphocytes enhances their antitumor response by making them more resistant to the acidic TME.

TCR-induced FOXP3 expression by CD8+ T cells impairs their anti-tumor activity.

Adoptive cell transfer therapy using CD8+ T lymphocytes showed promising results eradicating metastatic malignancies. However, several regulatory mechanisms limit its efficacy. We studied the role of the expression of the transcription factor FOXP3 on CD8+ T cell function and anti-tumor immunity. Here we show that suboptimal T cell receptor stimulation of CD8+ T cells upregulates FOXP3 in vitro. Similarly, CD8 T cells transferred into tumor-bearing mice upregulate FOXP3 in vivo. Cell-intrinsic loss of FOXP3 by CD8+ T cells resulted in improved functionality after TCR stimulation and better antitumor responses in vivo. Inhibition of the FOXP3/NFAT interaction likewise improved CD8+ T cell functionality. Transcriptomic analysis of cells after TCR stimulation revealed an enrichment of genes implicated in the response to IFN-γ, IFN-α, inflammatory response, IL-6/JAK/STAT, G2M checkpoint and IL-2/STAT signaling in FOXP3-deficient CD8+ T cells with respect to FOXP3-wt CD8+ T cells. Our results suggest that transient expression of FOXP3 by CD8+ T cells in the tumor microenvironment restrains their anti-tumor activity, with clear implications for improving T cell responses during immunotherapy.

Gemcitabine-mediated depletion of immunosuppressive dendritic cells enhances the efficacy of therapeutic vaccination.

Vaccination using optimized strategies may increase response rates to immune checkpoint inhibitors (ICI) in some tumors. To enhance vaccine potency and improve thus responses to ICI, we analyzed the gene expression profile of an immunosuppressive dendritic cell (DC) population induced during vaccination, with the goal of identifying druggable inhibitory mechanisms. RNAseq studies revealed targetable genes, but their inhibition did not result in improved vaccines. However, we proved that immunosuppressive DC had a monocytic origin. Thus, monocyte depletion by gemcitabine administration reduced the generation of these DC and increased vaccine-induced immunity, which rejected about 20% of LLC-OVA and B16-OVA tumors, which are non-responders to anti-PD-1. This improved efficacy was associated with higher tumor T-cell infiltration and overexpression of PD-1/PD-L1. Therefore, the combination of vaccine + gemcitabine with anti-PD-1 was superior to anti-PD-1 monotherapy in both models. B16-OVA tumors benefited from a synergistic effect, reaching 75% of tumor rejection, but higher levels of exhausted T-cells in LLC-OVA tumors co-expressing PD-1, LAG3 and TIM3 precluded similar levels of efficacy. Our results indicate that gemcitabine is a suitable combination therapy with vaccines aimed at enhancing PD-1 therapies by targeting vaccine-induced immunosuppressive DC.

Targeting the extra domain A of fibronectin for cancer therapy with CAR-T cells.

BACKGROUND: One of the main difficulties of adoptive cell therapies with chimeric antigen receptor (CAR)-T cells in solid tumors is the identification of specific target antigens. The tumor microenvironment can present suitable antigens for CAR design, even though they are not expressed by the tumor cells. We have generated a CAR specific for the splice variant extra domain A (EDA) of fibronectin, which is highly expressed in the tumor stroma of many types of tumors but not in healthy tissues. METHODS: EDA expression was explored in RNA-seq data from different human tumor types and by immunohistochemistry in paraffin-embedded tumor biopsies. Murine and human anti-EDA CAR-T cells were prepared using recombinant retro/lentiviruses, respectively. The functionality of EDA CAR-T cells was measured in vitro in response to antigen stimulation. The antitumor activity of EDA CAR-T cells was measured in vivo in C57BL/6 mice challenged with PM299L-EDA hepatocarcinoma cell line, in 129Sv mice-bearing F9 teratocarcinoma and in NSG mice injected with the human hepatocarcinoma cell line PLC. RESULTS: EDA CAR-T cells recognized and killed EDA-expressing tumor cell lines in vitro and rejected EDA-expressing tumors in immunocompetent mice. Notably, EDA CAR-T cells showed an antitumor effect in mice injected with EDA-negative tumor cells lines when the tumor stroma or the basement membrane of tumor endothelial cells express EDA. Thus, EDA CAR-T administration delayed tumor growth in immunocompetent 129Sv mice challenged with teratocarcinoma cell line F9. EDA CAR-T treatment exerted an antiangiogenic effect and significantly reduced gene signatures associated with epithelial-mesenchymal transition, collagen synthesis, extracellular matrix organization as well as IL-6-STAT5 and KRAS pathways. Importantly, the human version of EDA CAR, that includes the human 41BB and CD3ζ endodomains, exerted strong antitumor activity in NSG mice challenged with the human hepatocarcinoma cell line PLC, which expresses EDA in the tumor stroma and the endothelial vasculature. EDA CAR-T cells exhibited a tropism for EDA-expressing tumor tissue and no toxicity was observed in tumor bearing or in healthy mice. CONCLUSIONS: These results suggest that targeting the tumor-specific fibronectin splice variant EDA with CAR-T cells is feasible and offers a therapeutic option that is applicable to different types of cancer.

Intratumoral STING Agonist Injection Combined with Irreversible Electroporation Delays Tumor Growth in a Model of Hepatocarcinoma.

BACKGROUND/AIM: Irreversible electroporation (IRE) showed promising results for small-size tumors and very early cancers. However, further development is needed to evolve this procedure into a more efficient ablation technique for long-term control of tumor growth. In this work, we show that it is possible to increase the antitumor efficiency of IRE by simmultaneously injecting c-di-GMP, a STING agonist, intratumorally. MATERIALS AND METHODS: Intratumoral administration of c-di-GMP simultaneously to IRE was evaluated in murine models of melanona (B16.OVA) and hepatocellular carcinoma (PM299L). RESULTS: The combined therapy increased the number of tumor-infiltrating IFN-γ/TNF-α-producing CD4 and CD8 T cells and delayed tumor growth, as compared to the effect observed in groups treated with c-di-GMP or IRE alone. CONCLUSION: These results can lead to the development of a new therapeutic strategy for the treatment of cancer patients refractory to other therapies.

Alcohol exposure differentially effects anti-tumor immunity in females by altering dendritic cell function.

Dendritic cells (DCs) are a critical component of anti-tumor immunity due to their ability to induce a robust immune response to antigen (Ag). Alcohol was previously shown to reduce DC ability to present foreign Ag and promote pro-inflammatory responses in situations of infection and trauma. However the impact of alcohol exposure on generation of an anti-tumor response, especially in the context of generation of an immune vaccine has not been examined. In the clinic, DC vaccines are typically generated from autologous blood, therefore prior exposure to substances such as alcohol may be a critical factor to consider regarding the effectiveness in generating an immune response. In this study, we demonstrate for the first time that ethanol differentially affects DC and tumor Ag-specific T cell responses depending on sex. Signaling pathways were found to be differentially regulated in DC in females compared to males and these differences were exacerbated by ethanol treatment. DC from female mice treated with ethanol were unable to activate Ag-specific cytotoxic T cells (CTL) as shown by reduced expression of CD44, CD69, and decreased production of granzyme B and IFNγ. Furthermore, although FOXO3, an immune suppressive mediator of DC function, was found to be upregulated in DC from female mice, ethanol related suppression was independent of FOXO3. These findings demonstrate for the first time differential impacts of alcohol on the immune system of females compared to males and may be a critical consideration for determining the effectiveness of an immune based therapy for cancer in patients that consume alcohol.

Ccl22 Diverts T Regulatory Cells and Controls the Growth of Melanoma.

T regulatory cells (Treg) avert autoimmunity, but their increased levels in melanoma confer a poor prognosis. To explore the basis for Treg accumulation in melanoma, we evaluated chemokine expression in patients. A 5-fold increase was documented in the Treg chemoattractants CCL22 and CCL1 in melanoma-affected skin versus unaffected skin, as accompanied by infiltrating FoxP3+ T cells. In parallel, there was an approximately two-fold enhancement in expression of CCR4 in circulating Treg but not T effector cells. We hypothesized that redirecting Treg away from tumors might suppress autoimmune side effects caused by immune checkpoint therapeutics now used widely in the clinic. In assessing this hypothesis, we observed a marked increase in skin Treg in mice vaccinated with Ccl22, with repetitive vaccination sufficient to limit Treg accumulation and melanoma growth in the lungs of animals challenged by tumor cell injection, whether using a prevention or treatment protocol design. The observed change in Treg accumulation in this setting could not be explained by Treg conversion. Overall, our findings offered a preclinical proof of concept for the potential use of CCL22 delivered by local injection as a strategy to enhance the efficacious response to immune checkpoint therapy while suppressing its autoimmune side effects. Cancer Res; 76(21); 6230-40. ©2016 AACR.