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.

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.

The oncolytic adenovirus Delta-24-RGD in combination with ONC201 induces a potent antitumor response in pediatric high-grade and diffuse midline glioma models.

BACKGROUND: Pediatric high-grade gliomas (pHGGs), including diffuse midline gliomas (DMGs), are aggressive pediatric tumors with one of the poorest prognoses. Delta-24-RGD and ONC201 have shown promising efficacy as single agents for these tumors. However, the combination of both agents has not been evaluated. METHODS: The production of functional viruses was assessed by immunoblotting and replication assays. The antitumor effect was evaluated in a panel of human and murine pHGG and DMG cell lines. RNAseq, the seahorse stress test, mitochondrial DNA content, and γH2A.X immunofluorescence were used to perform mechanistic studies. Mouse models of both diseases were used to assess the efficacy of the combination in vivo. The tumor immune microenvironment was evaluated using flow cytometry, RNAseq and multiplexed immunofluorescence staining. RESULTS: The Delta-24-RGD/ONC201 combination did not affect the virus replication capability in human pHGG and DMG models in vitro. Cytotoxicity analysis showed that the combination treatment was either synergistic or additive. Mechanistically, the combination treatment increased nuclear DNA damage and maintained the metabolic perturbation and mitochondrial damage caused by each agent alone. Delta-24-RGD/ONC201 cotreatment extended the overall survival of mice implanted with human and murine pHGG and DMG cells, independent of H3 mutation status and location. Finally, combination treatment in murine DMG models revealed a reshaping of the tumor microenvironment to a proinflammatory phenotype. CONCLUSIONS: The Delta-24-RGD/ONC201 combination improved the efficacy compared to each agent alone in in vitro and in vivo models by potentiating nuclear DNA damage and in turn improving the antitumor (immune) response to each agent alone.

Intratumoral co-injection of NK cells and NKG2A-neutralizing monoclonal antibodies.

NK-cell reactivity against cancer is conceivably suppressed in the tumor microenvironment by the interaction of the inhibitory receptor NKG2A with the non-classical MHC-I molecules HLA-E in humans or Qa-1b in mice. We found that intratumoral delivery of NK cells attains significant therapeutic effects only if co-injected with anti-NKG2A and anti-Qa-1b blocking monoclonal antibodies against solid mouse tumor models. Such therapeutic activity was contingent on endogenous CD8 T cells and type-1 conventional dendritic cells (cDC1). Moreover, the anti-tumor effects were enhanced upon combination with systemic anti-PD-1 mAb treatment and achieved partial abscopal efficacy against distant non-injected tumors. In xenografted mice bearing HLA-E-expressing human cancer cells, intratumoral co-injection of activated allogeneic human NK cells and clinical-grade anti-NKG2A mAb (monalizumab) synergistically achieved therapeutic effects. In conclusion, these studies provide evidence for the clinical potential of intratumoral NK cell-based immunotherapies that exert their anti-tumor efficacy as a result of eliciting endogenous T-cell responses.

Regulatory T Cell Inhibition by P60 Combined with Adenoviral AFP Transduced Dendritic Cells for Immunotherapy of Hepatocellular Carcinoma.

BACKGROUND & AIMS: Vaccination with tumor-associated antigen-pulsed dendritic cells leads to specific T-cell response against hepatocellular carcinoma. However, clinical response has been shown to be limited. High regulatory T-cell count is associated with poor prognosis and seems to mediate immune tolerance in hepatocellular carcinoma. Forkhead box P3-peptide inhibitor P60 has been shown to specifically inhibit regulatory T-cell function in murine models. Aim of this study was to investigate whether P60 can improve the immune response induced by vaccination with adenovirus-transduced dendritic cells expressing alpha-fetoprotein in subcutaneous and orthotopic murine models for hepatocellular carcinoma. METHODS: Mice developing subcutaneous or orthotopic HCC received daily treatment with P60 starting at different tumor stages. Additionally, mice were vaccinated twice with dendritic cells expressing alpha-fetoprotein. RESULTS: In a preventive setting prior to tumor engraftment, vaccination with alpha-fetoprotein-expressing dendritic cells significantly decreased tumor growth in a subcutaneous model (p = .0256), but no further effects were achieved by addition of P60. However, P60 enhanced the antitumoral effect of a vaccination with alpha-fetoprotein-expressing dendritic cells in established subcutaneous and orthotopic hepatocellular carcinoma characterized by high Treg levels (p = .011). CONCLUSION: In this study, we showed that vaccination with alpha-fetoprotein-expressing dendritic cells in combination with a specific inhibition of regulatory T-cells by using P60 leads to synergistic tumor inhibition and prolonged survival. This emphasizes the importance of regulatory T-cells inhibition for obtaining an effective antitumoral immune response in hepatocellular carcinoma.

Targeting FOXP3 Tumor-Intrinsic Effects Using Adenoviral Vectors in Experimental Breast Cancer.

The regulatory T cell master transcription factor, Forkhead box P3 (Foxp3), has been detected in cancer cells; however, its role in breast tumor pathogenesis remains controversial. Here we assessed Foxp3 tumor intrinsic effects in experimental breast cancer using a Foxp3 binder peptide (P60) that impairs Foxp3 nuclear translocation. Cisplatin upregulated Foxp3 expression in HER2+ and triple-negative breast cancer (TNBC) cells. Foxp3 inhibition with P60 enhanced chemosensitivity and reduced cell survival and migration in human and murine breast tumor cells. We also developed an adenoviral vector encoding P60 (Ad.P60) that efficiently transduced breast tumor cells, reduced cell viability and migration, and improved the cytotoxic response to cisplatin. Conditioned medium from transduced breast tumor cells contained lower levels of IL-10 and improved the activation of splenic lymphocytes. Intratumoral administration of Ad.P60 in breast-tumor-bearing mice significantly reduced tumor infiltration of Tregs, delayed tumor growth, and inhibited the development of spontaneous lung metastases. Our results suggest that Foxp3 exerts protumoral intrinsic effects in breast cancer cells and that gene-therapy-mediated blockade of Foxp3 could constitute a therapeutic strategy to improve the response of these tumors to standard treatment.

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.

Erratum: Enhanced cross-recognition of SARS-CoV-2 Omicron variant by peptide vaccine-induced antibodies.

[This corrects the article DOI: 10.3389/fimmu.2022.1044025.].

Silver nanoparticles induce a non-immunogenic tumor cell death.

Immunogenic cell death (ICD) is a form of cell death characterized by the release of danger signals required to trigger an adaptive immune response against tumor-associated antigens. Silver nanoparticles (AgNP) display anti-proliferative and cytotoxic effects in tumor cells, but it has not been previously studied whether AgNP act as an ICD inductor. The present study evaluated the in vitro release of calreticulin as a damage-associated molecular pattern (DAMP) associated with the cytotoxicity of AgNP and their in vivo anti-cancer effects. In vitro, mouse CT26 colon carcinoma and MCA205 fibrosarcoma cells were exposed to AgNP and then cell proliferation, adhesion, and release of calreticulin were determined. The results indicated there were time- and concentration-related anti-proliferative effects of AgNP in both the CT26 and MCA205 lines. Concurrently, changes in cell adhesion were detected mainly in the CT26 cells. Regarding DAMP detection, a significant increase in calreticulin was observed only in CT26 cells treated with doxorubicin and AgNP; however, no differences were found in the MCA205 cells. In vivo, the survival and growth of subcutaneous tumors were monitored after vaccination of mice with cell debris from tumor cells treated with AgNP or after intra-tumoral administration of AgNP to established tumors. Consequently, anti-tumoral prophylactic immunization with AgNP-dead cells failed to protect mice from tumor re-challenge; intra-tumor injection of AgNP did not induce a significant effect. In conclusion, there was a noticeable anti-tumoral effect of AgNP in vitro in both CT26 and MCA205 cell lines, accompanied by the release of calreticulin in CT26 cells. In vivo, immunization with cell debris derived from AgNP-treated tumor cells failed to induce a protective immune response in the cancer model mice. Clearly, further research is needed to determine if one could combine AgNP with other ICD inducers to improve the anti-tumor effect of these nanoparticles in vivo.

FOXP3 expression diversifies the metabolic capacity and enhances the efficacy of CD8 T cells in adoptive immunotherapy of melanoma.

Regulatory T cells overwhelm conventional T cells in the tumor microenvironment (TME) thanks to a FOXP3-driven metabolic program that allows them to engage different metabolic pathways. Using a melanoma model of adoptive T cell therapy (ACT), we show that FOXP3 overexpression in mature CD8 T cells improved their antitumor efficacy, favoring their tumor recruitment, proliferation, and cytotoxicity. FOXP3-overexpressing (Foxp3UP) CD8 T cells exhibited features of tissue-resident memory-like and effector T cells, but not suppressor activity. Transcriptomic analysis of tumor-infiltrating Foxp3UP CD8 T cells showed positive enrichment in a wide variety of metabolic pathways, such as glycolysis, fatty acid (FA) metabolism, and oxidative phosphorylation (OXPHOS). Intratumoral Foxp3UP CD8 T cells exhibited an enhanced capacity for glucose and FA uptake as well as accumulation of intracellular lipids. Interestingly, Foxp3UP CD8 T cells compensated for the loss of mitochondrial respiration-driven ATP production by activating aerobic glycolysis. Moreover, in limiting nutrient conditions these cells engaged FA oxidation to drive OXPHOS for their energy demands. Importantly, their ability to couple glycolysis and OXPHOS allowed them to sustain proliferation under glucose restriction. Our findings demonstrate a hitherto unknown role for FOXP3 in the adaptation of CD8 T cells to TME that may enhance their efficacy in ACT.

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.

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.

Inhibiting Histone and DNA Methylation Improves Cancer Vaccination in an Experimental Model of Melanoma.

Immunotherapy has improved the treatment of malignant skin cancer of the melanoma type, yet overall clinical response rates remain low. Combination therapies could be key to meet this cogent medical need. Because epigenetic hallmarks represent promising combination therapy targets, we studied the immunogenic potential of a dual inhibitor of histone methyltransferase G9a and DNA methyltransferases (DNMTs) in the preclinical B16-OVA melanoma model. Making use of tumor transcriptomic and functional analyses, methylation-targeted epigenetic reprogramming was shown to induce tumor cell cycle arrest and apoptosis in vitro coinciding with transient tumor growth delay and an IFN-I response in immune-competent mice. In consideration of a potential impact on immune cells, the drug was shown not to interfere with dendritic cell maturation or T-cell activation in vitro. Notably, the drug promoted dendritic cell and, to a lesser extent, T-cell infiltration in vivo, yet failed to sensitize tumor cells to programmed cell death-1 inhibition. Instead, it increased therapeutic efficacy of TCR-redirected T cell and dendritic cell vaccination, jointly increasing overall survival of B16-OVA tumor-bearing mice. The reported data confirm the prospect of methylation-targeted epigenetic reprogramming in melanoma and sustain dual G9a and DNMT inhibition as a strategy to tip the cancer-immune set-point towards responsiveness to active and adoptive vaccination against melanoma.

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.

Enhanced cross-recognition of SARS-CoV-2 Omicron variant by peptide vaccine-induced antibodies.

Current vaccines against SARS-CoV-2, based on the original Wuhan sequence, induce antibodies with different degrees of cross-recognition of new viral variants of concern. Despite potent responses generated in vaccinated and infected individuals, the Omicron (B.1.1.529) variant causes breakthrough infections, facilitating viral transmission. We previously reported a vaccine based on a cyclic peptide containing the 446-488 S1 sequence (446-488cc) of the SARS-CoV-2 spike (S) protein from Wuhan isolate. To provide the best immunity against Omicron, here we compared Omicron-specific immunity induced by a Wuhan-based 446-488cc peptide, by a Wuhan-based recombinant receptor-binding domain (RBD) vaccine and by a new 446-488cc peptide vaccine based on the Omicron sequence. Antibodies induced by Wuhan peptide 446-488cc in three murine strains not only recognized the Wuhan and Omicron 446-488 peptides similarly, but also Wuhan and Omicron RBD protein variants. By contrast, antibodies induced by the Wuhan recombinant RBD vaccine showed a much poorer cross-reactivity for the Omicron RBD despite similar recognition of Wuhan and Omicron peptide variants. Finally, although the Omicron-based 446-488cc peptide vaccine was poorly immunogenic in mice due to the loss of T cell epitopes, co-immunization with Omicron peptide 446-488cc and exogenous T cell epitopes induced strong cross-reactive antibodies that neutralized Omicron SARS-CoV-2 virus. Since mutations occurring within this sequence do not alter T cell epitopes in humans, these results indicate the robust immunogenicity of 446-488cc-based peptide vaccines that induce antibodies with a high cross-recognition capacity against Omicron, and suggest that this sequence could be included in future vaccines targeting the Omicron variant.

Olfactory Characterization and Training in Older Adults: Protocol Study.

The aim of this article is to present the research protocol for a prospective cohort study that will assess the olfactory function and the effect of an intervention based on olfactory training in healthy very old adults (≥75 years old). A convenience sample of 180 older people (50% female) will be recruited in three different environments: hospitalized control group (CH) with stable acute illness (n = 60); ambulatory control group (CA) of community-based living (n = 60); and an experimental odor training group (EOT) from nursing homes (n = 60). The odor training (OT) intervention will last 12 weeks. All the volunteers will be assessed at baseline; CA and EOT groups will also be assessed after 12 weeks. The primary end point will be change in olfactory capacity from baseline to 12 weeks period of intervention or control. The intervention effects will be assessed with the overall score achieved in Sniffin Sticks Test (SST) - Threshold, Discrimination, and Identification (TDI) extended version. Secondary end points will be changes in cognitive tasks, quality of life, mood, immune status, and functional capacity. All these measurements will be complemented with an immune fitness characterization and a deep proteome profiling of the olfactory epithelium (OE) cultured ex vivo. The current study will provide additional evidence to support the implementation of olfactory precision medicine and the development of immunomodulatory nasal therapies based on non-invasive procedures. The proposed intervention will also intend to increase the knowledge about the olfactory function in very elderly people, improve function and quality of life, and promote the recovery of the health.

Epitope spreading driven by the joint action of CART cells and pharmacological STING stimulation counteracts tumor escape via antigen-loss variants.

BACKGROUND: Target antigen (Ag) loss has emerged as a major cause of relapse after chimeric antigen receptor T (CART)-cell therapy. We reasoned that the combination of CART cells, with the consequent tumor debulking and release of Ags, together with an immunomodulatory agent, such as the stimulator of interferon gene ligand (STING-L) 2'3'-cyclic GMP-AMP (2'3'-cGAMP), may facilitate the activation of an endogenous response to secondary tumor Ags able to counteract this tumor escape mechanism. METHODS: Mice bearing B16-derived tumors expressing prostate-specific membrane Ag or gp75 were treated systemically with cognate CART cells followed by intratumoral injections of 2'3'-cGAMP. We studied the target Ag inmunoediting by CART cells and the effect of the CART/STING-L combination on the control of STING-L-treated and STING-L-non-treated tumors and on the endogenous antitumor T-cell response. The role of Batf3-dependent dendritic cells (DCs), stimulator of interferon gene (STING) signaling and perforin (Perf)-mediated killing in the efficacy of the combination were analyzed. RESULTS: Using an immune-competent solid tumor model, we showed that CART cells led to the emergence of tumor cells that lose the target Ag, recreating the cancer immunoediting effect of CART-cell therapy. In this setting, the CART/STING-L combination, but not the monotherapy with CART cells or STING-L, restrained tumor progression and enhanced overall survival, showing abscopal effects on distal STING-L-non-treated tumors. Interestingly, a secondary immune response against non-chimeric antigen receptor-targeted Ags (epitope spreading), as determined by major histocompatibility complex-I-tetramer staining, was fostered and its intensity correlated with the efficacy of the combination. This was consistent with the oligoclonal expansion of host T cells, as revealed by in-depth T-cell receptor repertoire analysis. Moreover, only in the combination group did the activation of endogenous T cells translate into a systemic antitumor response. Importantly, the epitope spreading and the antitumor effects of the combination were fully dependent on host STING signaling and Batf3-dependent DCs, and were partially dependent on Perf release by CART cells. Interestingly, the efficacy of the CART/STING-L treatment also depended on STING signaling in CART cells. CONCLUSIONS: Our data show that 2'3'-cGAMP is a suitable adjuvant to combine with CART-cell therapy, allowing the induction of an endogenous T-cell response that prevents the outgrowth of Ag-loss tumor variants.

Preclinical evaluation of a synthetic peptide vaccine against SARS-CoV-2 inducing multiepitopic and cross-reactive humoral neutralizing and cellular CD4 and CD8 responses.

Identification of relevant epitopes is crucial for the development of subunit peptide vaccines inducing neutralizing and cellular immunity against SARS-CoV-2. Our aim was the characterization of epitopes in the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein to generate a peptide vaccine. Epitope mapping using a panel of 10 amino acid overlapped 15-mer peptides covering region 401-515 from RBD did not identify linear epitopes when tested with sera from infected individuals or from RBD-immunized mice. However, immunization of mice with these 15-mer peptides identified four peptides located at region 446-480 that induced antibodies recognizing the peptides and RBD/S1 proteins. Immunization with peptide 446-480 from S protein formulated with Freund's adjuvant or with CpG oligodeoxinucleotide/Alum induced polyepitopic antibody responses in BALB/c and C56BL/6J mice, recognizing RBD (titres of 3 × 104-3 × 105, depending on the adjuvant) and displaying neutralizing capacity (80-95% inhibition capacity; p < 0.05) against SARS-CoV-2. Murine CD4 and CD8T-cell epitopes were identified in region 446-480 and vaccination experiments using HLA transgenic mice suggested the presence of multiple human T-cell epitopes. Antibodies induced by peptide 446-480 showed broad recognition of S proteins and S-derived peptides belonging to SARS-CoV-2 variants of concern. Importantly, vaccination with peptide 446-480 or with a cyclic version of peptide 446-488 containing a disulphide bridge between cysteines 480 and 488, protected humanized K18-hACE2 mice from a lethal dose of SARS-CoV-2 (62.5 and 75% of protection; p < 0.01 and p < 0.001, respectively). This region could be the basis for a peptide vaccine or other vaccine platforms against Covid-19.

Dual activity of PD-L1 targeted Doxorubicin immunoliposomes promoted an enhanced efficacy of the antitumor immune response in melanoma murine model.

BACKGROUND: The immunomodulation of the antitumor response driven by immunocheckpoint inhibitors (ICIs) such as PD-L1 (Programmed Death Ligand-1) monoclonal antibody (α-PD-L1) have shown relevant clinical outcomes in a subset of patients. This fact has led to the search for rational combinations with other therapeutic agents such as Doxorubicin (Dox), which cytotoxicity involves an immune activation that may enhance ICI response. Therefore, this study aims to evaluate the combination of chemotherapy and ICI by developing Dox Immunoliposomes functionalized with monovalent-variable fragments (Fab') of α-PD-L1. RESULTS: Immunoliposomes were assayed in vitro and in vivo in a B16 OVA melanoma murine cell line over-expressing PD-L1. Here, immune system activation in tumor, spleen and lymph nodes, together with the antitumor efficacy were evaluated. Results showed that immunoliposomes bound specifically to PD-L1+ cells, yielding higher cell interaction and Dox internalization, and decreasing up to 30-fold the IC50, compared to conventional liposomes. This mechanism supported a higher in vivo response. Indeed, immunoliposomes promoted full tumor regression in 20% of mice and increased in 1 month the survival rate. This formulation was the only treatment able to induce significant (p < 0.01) increase of activated tumor specific cytotoxic T lymphocytes at the tumor site. CONCLUSION: PD-L1 targeted liposomes encapsulating Dox have proved to be a rational combination able to enhance the modulation of the immune system by blocking PD-L1 and selectively internalizing Dox, thus successfully providing a dual activity offered by both, chemo and immune therapeutic strategies.