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To investigate whether different restoration designs, overlay types, and full crowns in posterior teeth have similarly acceptable marginal sealing and quality.For Part 1 of the present study (investigation of fracture resistance), 70 extracted molars were divided into five groups (N = 14), prepared with four different posterior indirect adhesive restoration (PIAR) overlay design types, according to the adhesthetics classification. The groups were: 1. Butt Joint; 2. Full Bevel; 3. Shoulder; 4. Full Crown; 5. Sound Tooth. For Part 2 of the study (present article; marginal quality), there was no group 5, and only 56 of the 70 extracted molars were used. Seven expert dentists performed all the preparation and cementation phases with codified protocols. A CAD/CAM workflow was used to realize the 56 monolithic lithium disilicate restorations. The samples were tested with thermomechanical loading (TML) and the marginal quality evaluated. The data relating to fracture resistance are presented in Part 1 of this study (Int J Esthet Dent 2021;16:2-17).In terms of marginal quality after TML, within the limitations of the present study, in molar teeth (without endodontic treatments) restored with different monolithic ceramic lithium disilicate PIAR designs, it is possible to present the following conclusions.

Introduction: Tertiary Lymphoid Structures (TLS) are highly organized ectopic lymphoid structures found in inflamed or tumor tissues, acting as sites of lymphoid recruitment and immune activation. A high TLS density within the tumor is commonly associated with an increased prognostic effect of TILs and with an improved disease free survival and overall survival for patients. However, the existence of conflicting studies suggest that multiple TLS features should be taken into account when assessing their prognostic value, such as their location, cellular composition, maturation stage and spatial organisation, as those may affect their functionalities. Methods: With the aim of gaining insights into TLS biology and evaluating the prognostic role of TLS in Non-Small Cell Lung Carcinoma (NSCLC) according to their multiple features, we developed a TLS multiplex immunofluorescent (mIF) panel that includes T cell (CD3, CD8), B cell (CD20), Follicular Dendritic cell (CD21, CD23) and mature dendritic cell (DC-LAMP) markers. We deployed this panel across a cohort of primary tumors from NSCLC patients (n=408) and established a mIF image analysis workstream to assess the status and spatial location of each cell within the tissue. A H&E staining of the same tissue section was performed to evaluate mIF spatial data in relation to the tumor context. Additional multi-omics assessments were conducted across the same cohort including; whole exome sequencing, NanoString transcriptomics, and immunohistochemistry (e.g. PD-L1, FOXP3). We have leveraged clinical metadata, including demographics (e.g. age, sex, smoking status) and clinical risk factors (e.g. stage, grade, Standard of Care treatment) with clinical follow up (e.g. OS, PFS) for prevalence analysis, novel biomarker identification, and survival association. Results: Assessment of the prevalence of each cell phenotype within the tumor tissue and TLS (tumor centre vs invasive margin; tumor epithelium vs stroma), the distance between each cell type, and the distance of non-TLS immune cells to the closest TLS will be described, demonstrating the different types of lymphoid aggregates and TLS and their functional status. An integrative analysis combining these spatial biology data with multi-omics and clinical data will be presented evaluating the prognostic value of TLS composition, maturation status and spatial organization, in correlation with additional biomarkers and clinical characteristics. Conclusion: This exploratory study using cutting-edge technologies enables us to better understand how TLS orchestrate an organised anti-tumour response, defining TLS spatial biomarker signatures, TLS gene signatures, and TLS features associated with NSCLC patient outcomes to evaluate in the clinic. Citation Format: Julie Berthe, Felix Segerer, Emily C. Jennings, Alma Andoni, Marco Testori, Megha Saraiya, Miljenka Vuko, Harald Hessel, Andreas Spitzmüller, Mari Heininen-Brown, Jorge Blando, Felicia Ng, Emma Jones, Sophie Willis, Michael Surace, Rieneke van de Ven, Tanja De Gruijl, Helen Angell. A multi-modal analysis approach leveraging multiplexed spatial phenotyping and multi-omics analysis to better understand the prognostic value of tertiary lymphoid structures in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1711.

BackgroundTertiary Lymphoid Structures (TLS) are highly organized ectopic lymphoid structures found in inflamed or tumor tissues, acting as sites of lymphoid recruitment and immune activation. A high TLS density within the tumor is commonly associated with an increased prognostic effect of TILs and with an improved disease free survival and overall survival for patients.1 However, the existence of conflicting studies suggest that multiple TLS features should be taken into account when assessing their prognostic value, such as their location, cellular composition, maturation stage and spatial organisation, as those may affect their functionalities.2MethodsWith the aim of gaining insights into TLS biology and evaluating the prognostic role of TLS in Non-Small Cell Lung Carcinoma according to their multiple features, we developed a TLS multiplex immunofluorescent (mIF) panel that includes T cells (CD3, CD8), B cells (CD20), Follicular Dendritic cells (CD21, CD23) and mature dendritic cells (DC-LAMP) markers. We deployed this panel across a cohort of primary tumors from NSCLC patients (n=408) and established a mIF image analysis workstream to assess the status and spatial location of each cell within the tissue. A H&E staining of the same tissue section was performed to evaluate mIF spatial data in relation to the tumor context. Additional multi-omics assessments were conducted across the same cohort including; whole exome sequencing, NanoString transcriptomics, and immunohistochemistry (e.g. PD-L1, FOXP3, NKp46, LKB1, CTLA4). We have leveraged clinical metadata, including demographics (e.g. age, sex, smoking status) and clinical risk factors (e.g. stage, grade, Standard of Care treatment) with clinical follow up (e.g. OS, PFS) for prevalence analysis, novel biomarker identification, and survival association.ResultsAssessment of the prevalence of each cell phenotype within the tumor tissue and TLS, the cell-cell interactions, the distance between each cell type, and the distance of non-TLS immune cells to the closest TLS will be described, demonstrating the different types of lymphoid aggregates and TLS and their functional status. An integrative analysis combining spatial biology data with multi-omics and clinical data will be presented evaluating the prognostic value of TLS composition, maturation status and spatial organization, in correlation with additional biomarkers and clinical characteristics.ConclusionsThis exploratory study using cutting-edge technologies enables us to better understand how TLS orchestrate an organised anti-tumour response, defining TLS spatial biomarker signatures, TLS gene signatures, and TLS features associated with patient outcomes to evaluate in the clinic.ReferencesMarie-Caroline Dieu-Nosjean, Jérémy Goc, Nicolas A Giraldo, Catherine Sautès-Fridman, Wolf Herman Fridman. Tertiary lymphoid structures in cancer and beyond. Trends Immunol 2014;35(11):571–580.Catherine Sautès-Fridman, Florent Petitprez, Julien Calderaro, Wolf Herman Fridman. Tertiary lymphoid structures in the era of cancer immunotherapy. Nat Rev Cancer 2019;19(6):307–325.Ethics ApprovalThe study was approved by AstraZeneca.

Virus-based vaccines and appropriate costimulation potently enhance antigen-specific T cell immunity against cancer. Here we report the use of recombinant modified vaccinia virus Ankara (rMVA) encoding costimulatory CD40L against solid tumors. Therapeutic treatment with rMVA-CD40L-expressing tumor-associated antigens results in the control of established tumors. The expansion of tumor-specific cytotoxic CD8+ T cells is essential for the therapeutic antitumor effects. Strikingly, rMVA-CD40L also induces strong natural killer (NK) cell activation and expansion. Moreover, the combination of rMVA-CD40L and tumor-targeting antibodies results in increased therapeutic antitumor efficacy relying on the presence of Fc receptor and NK cells. We describe a translationally relevant therapeutic synergy between systemic viral vaccination and CD40L costimulation. We show strengthened antitumor immune responses when both rMVA-CD40L-induced innate and adaptive immune mechanisms are exploited by combination with tumor-targeting antibodies. This immunotherapeutic approach could translate into clinical cancer therapies where tumor-targeting antibodies are employed., CD40 agonists have been investigated as a strategy to awaken the immune system against cancers. Here, the authors use a virus encoding CD40L and tumour-associated antigens to enhance innate and adaptive immunity that together with tumour targeting antibodies controls the growth of tumours in mice.

Bacterial flagellin enhances innate and adaptive immune responses and is considered a promising adjuvant for the development of vaccines against infectious diseases and cancer. Antigen-presenting cells recognize flagellin with the extracellular TLR5 and the intracellular NLRC4 inflammasome-mediated pathway. The detailed cooperation of these innate pathways in the induction of the adaptive immune response following intranasal (i.n.) administration of a recombinant modified vaccinia virus Ankara (rMVA) vaccine encoding flagellin (rMVA-flagellin) is not known. rMVA-flagellin induced enhanced secretion of mucosal IL-1β and TNF-α resulting in elevated CTL and IgG2c antibody responses. Importantly, mucosal IgA responses were also significantly enhanced in both bronchoalveolar (BAL) and intestinal lavages accompanied by the increased migration of CD8+ T cells to the mesenteric lymph nodes (MLN). Nlrc4−/− rMVA-flagellin-immunized mice failed to enhance pulmonary CTL responses, IgG2c was lower, and IgA levels in the BAL or intestinal lavages were similar as those of control mice. Our results show the favorable adjuvant effect of rMVA-flagellin in the lung as well as the intestinal mucosa following i.n. administration with NLRC4 as the essential driver of this promising mucosal vaccine concept.

Virus-based vaccines and appropriate costimulation enhance potent antigen-specific T cell immunity against cancer. However, the tumor microenvironment exerts intrinsic and extrinsic mechanisms to evade tumor destruction. In the present study we exploit both innate and adaptive immune responses triggered by a novel recombinant modified vaccinia virus Ankara (rMVA) encoding costimulatory CD40L against solid tumors in combination regimes to overcome tumor-induced resistance to immunotherapy. Therapeutic treatment with rMVA-CD40L resulted in strong antitumor effects in unrelated established tumor models. Tumor infiltration was composed of non-exhausted, antigen-specific CD8+ T cells with proliferative capacity after rMVA-CD40L immunization. Strikingly, this antitumor effect was not entirely dependent on cross-presenting CD8α+ DC -induced CD8+ T cell expansion, as experiments with MVA encoding for T. gondii profilin (TLR11 ligand) and using Batf3-/- tumor bearers depicted. Indeed, rMVA-CD40L-induced tumor control did not depend on cytosolic DNA sensor STING. Interestingly, rMVA-CD40L induced strong NK cell activation and thereby potent Antibody Dependent Cell Cytotoxicity (ADCC) against Tumor-Associated Antigen (TAA) targeting antibodies. Hence, the combination of TAA targeting antibodies and rMVA-CD40L resulted in increased therapeutic antitumor efficacy. We describe a novel and translationally relevant therapeutic synergy between viral vaccination and CD40L costimulation. We connect CD40 ligation to cross-presenting CD8α+ DC -mediated expansion of non-exhausted CD8+ T cells in the tumor microenvironment. Taking advantage from intrinsic MVA-induced NK cell activation and further improved NK cell function by CD40 ligation, we show strengthened antitumor immune responses when both rMVA-CD40L-induced innate and adaptive immune mechanisms are exploited by combining immunotherapeutic regimes. This finding has a direct potential impact in clinical trials where TAA targeting antibodies are currently under evaluation. Citation Format: Jose Medina-Echeverz, Maria Hinterberger, Marco Testori, Marlene Geiger, Raphael Giessel, Paul Chaplin, Hubertus Hochrein, Henning Lauterbach. A novel rMVA combination immunotherapy triggers potent innate and adaptive immune responses against established tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 727.

Background A new class of antiretrovirals, AntiViral-HyperActivation Limiting Therapeutics (AV-HALTs), has been proposed as a disease-modifying therapy to both reduce Human Immunodeficiency Virus Type 1 (HIV-1) RNA levels and the excessive immune activation now recognized as the major driver of not only the continual loss of CD4+ T cells and progression to Acquired Immunodeficiency Syndrome (AIDS), but also of the emergence of both AIDS-defining and non-AIDS events that negatively impact upon morbidity and mortality despite successful (ie, fully suppressive) therapy. VS411, the first-in-class AV-HALT, combined low-dose, slow-release didanosine with low-dose hydroxycarbamide to accomplish both objectives with a favorable toxicity profile during short-term administration. Five dose combinations were administered as VS411 to test the AV-HALT Proof-of-Concept in HIV-1-infected subjects. Methods Multinational, double-blind, 28-day Phase 2a dose-ranging Proof-of-Concept study of antiviral activity, immunological parameters, safety, and genotypic resistance in 58 evaluable antiretroviral-naïve HIV-1-infected adults. Randomization and allocation to study arms were carried out by a central computer system. Results were analyzed by ANOVA, Kruskal-Wallis, ANCOVA, and two-tailed paired t tests. Results VS411 was well-tolerated, produced significant reductions of HIV-1 RNA levels, increased CD4+ T cell counts, and led to significant, rapid, unprecedented reductions of immune activation markers after 28 days despite incomplete viral suppression and without inhibiting HIV-1-specific immune responses. The didanosine 200 mg/HC 900 mg once-daily formulation demonstrated the greatest antiviral efficacy (HIV-1 RNA: −1.47 log10 copies/mL; CD4+ T cell count: +135 cells/mm3) and fewest adverse events. Conclusions VS411 successfully established the Proof-of-Concept that AV-HALTs can combine antiviral efficacy with rapid, potentially beneficial reductions in the excessive immune system activation associated with HIV-1 disease. Rapid reductions in markers of immune system hyperactivation and cellular proliferation were obtained despite the fact that VS411 did not attain maximal suppression of HIV RNA, suggesting this effect was due to the HALT component. Trial Registration ITEudraCT 2007-002460-98