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-1 beta 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 APP(NL-G-F/NL-G-F) Alzheimer ' s mouse model. In all cases, the improvement in learning capacity was associated with a downregulation of IL-1b 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 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.

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-gamma, IFN-alpha, 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.

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 CD3 zeta 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.

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-gamma/TNF-alpha-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.