In chronic hepatitis B virus infection (cHBV), T-cell responses are skewed. This prevents the elimination of HBV-infected hepatocytes, contributes to hepatocellular carcinoma development, and limits the efficacy of T-cell therapies. To counteract the immune checkpoint PD-1/PD-L1-interaction in adoptive T-cell therapy, we developed PD-1-based immunoswitch receptors that convert PD-L1 engagement into co-stimulation. We designed immunoswitch receptors linking a PD-1 ectodomain to intracellular CD28, 4-1BB, or OX40 signaling domains and expressed them in HBV-specific chimeric antigen receptor (CAR)- and T-cell receptor-engineered T cells. T-cell activity was assessed in antigen- and PD-L1-dependent co-cultures, transcription factor reporter systems, and an HBV carrier mouse model. Second-generation S-CAR functionality was not improved by signal stacking of additional co-stimulatory domains, resulting in a dysfunctional activation state rather than an additive benefit. In contrast, modular delivery of co-stimulation through PD-1-based immunoswitch receptors enhanced antigen sensitivity, functionality, and cytotoxicity of engineered T cells. PD-L1 supplied in trans was sufficient to support on-target functionality of engineered T cells by immunoswitch receptor co-stimulation. PD-1_4-1BB proved superior at inducing sustained NF-κB signaling and reducing exhaustion-associated pathways. In vivo, PD-1_4-1BB improved T-cell persistence and decreased TOX upregulation. PD-1-based immunoswitch receptors offer a modular strategy to strengthen engineered T-cell responses against HBV antigen-expressing cells in the liver's tolerizing environment. This ligand-driven approach enables precise tuning of co-stimulation, generating a versatile platform for enhancing adoptive T-cell therapies in chronic infections and cancer.

Abstract Mounting evidence indicates that T cells can operate in an innate-like mode challenging the classical description of T cells as strictly adaptive immune effectors. T cells can engage innate pattern recognition receptors to mount rapid but antigen-nonspecific responses to infection or cellular stress. This study observed that CD8+ T cells, and to a lesser extent also CD4+ T cells, responded to viral proteins in the mouse lung quickly in an innate-like fashion. We employed intravital lung microscopy to visualize infiltration of CD8+ T cells into the lung following intratracheal instillation of the SARS-CoV-2 envelope (E)-protein. Here, we demonstrate acute recruitment of CD8+ from the pulmonary microcirculation into the lung as early as 4 and 24 hours after (E)-protein instillation. The acute infiltration of CD8+ T cells was not observed in Tlr2 − / − mice. Immunohistochemistry analysis of mouse lungs revealed T cell accumulation in nodular inflammatory foci (NIF) of the lung at perivascular regions and around large airways. Stimulating spleen-derived CD8+ T cells from wild-type mice with (E)-protein ex vivo in combination with cytokines or TCR agonists significantly upregulated CD69 and activated secretion of interferon (IFN)γ which was not observed with CD8+ T cells isolated from Tlr2 − / − mice. These findings indicate rapid bystander activation of CD8+ T cells by the SARS-CoV-2 envelope (E)-protein that depends on (E)-protein sensing by TLR2. This innate-like CD8+ T cell response to SARS-CoV-2 (E)-protein may offer novel opportunities for diagnostic and therapeutic development, warranting further investigation.

Immunotherapy with immune checkpoint inhibitors (ICI) has substantially improved the treatment of advanced renal cell carcinoma (aRCC). Since treatment effects vary among patients, identifying biomarkers to predict response is crucial for optimizing clinical management. Expression of programmed death ligand 1 (PD-L1) is associated with better response to ICI treatment, but is not a valid biomarker. To advance patient stratification for aRCC, we analyzed molecular and clinical data from the JAVELIN Renal 101 (n=726) and IMmotion151 (n=823) trials, both of which compared a combination of PD-L1 inhibitors and anti-angiogenic treatments with sunitinib monotherapy. In JAVELIN Renal 101, we found correlations between genes and progression-free survival (PFS) in PD-L1-positive tumors treated with sunitinib. These associations were independent of variations in the proportion of PD-L1-positive immune cells present in the tumors. Clustering of PD-L1-positive tumors based on PFS-related genes revealed an IMmune CHeckpoint Inhibitor-responsive Phenotype (IMCHIP). In PD-L1-positive tumors with IMCHIP, combined therapy with ICI led to significantly longer PFS relative to sunitinib (13.3 months vs. 4.4 months median PFS), while in PD-L1-positive tumors without IMCHIP, PFS was comparable for both treatments (11.1 months). By de-correlating gene expression measurements from PD-L1 expression, the clustering could be extended to PD-L1-negative tumors resulting in two groups (aRCC1/2) that were independent of PD-L1 status. Stratification based on aRCC1/2 and PD-L1 status allowed treatment effects to be categorized into four groups. A significant difference in PFS between treatments arms was only observed in patients with tumors of type aRCC2/PD-L1-positive, which corresponded to the IMCHIP profile and covered 32.4% of JAVELIN Renal 101. These findings could be validated in the IMmotion151 trial, where IMCHIP represented 20% of the cohort. Compared to other biomarkers currently being investigated, the combination of aRCC1/2 with PD-L1 status exhibited the greatest potential as a predictive biomarker for patient selection, which was confirmed in both cohorts. The prognostic potential of the aRCC1/2 subdivision was evaluated in 476 clear cell RCC from TCGA. Here, aRCC2 was associated with significantly worse overall survival and cancer-specific survival in both univariate and multivariable analyses, accounting for age, sex, and clinicopathological parameters (TNM). In summary, categorization into aRCC1/2 proved to be a new risk stratification of aRCC which, in combination with PD-L1 status, enabled the retrospective identification of responders who benefited from combined therapy with ICI relative to sunitinib monotherapy. The analysis of IMmotion151 trial data utilized data generated by Genentech/Genentech Research and Early Development. Florian A. Büttner, Viktor Grünwald, Elfriede Noessner, Igor Tsaur, Jens Bedke, Matthias Schwab, Elke Schaeffeler. A PD-L1-independent phenotype uncovers responders to combined therapy with immune checkpoint inhibitors in kidney cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7155.

We explored the dynamic changes of major leukocyte subsets during definitive treatment of patients with inoperable stage II/III NSCLC lung cancer and correlated it to survival to identify subpopulations associated with maximal patient benefit. We analyzed peripheral blood of 20 patients, either treated with thoracic radiotherapy (RT), concurrent chemo-radiotherapy (cCRT), or cCRT with additional immune-checkpoint inhibition therapy. Peripheral blood of 20 patients was collected at 9 timepoints before, during, and up to 1 year post treatment and analyzed by multi-color flow cytometry. Statistical analysis was conducted for leukocyte subpopulations, IL-6, progression-free survival (PFS) and overall survival (OS). Increase of absolute lymphocyte counts (ALC) after the end of RT until 6 months thereafter was a predictor of PFS. Baseline lymphocyte counts showed no significant correlation to PFS or OS. Early recovery of absolute counts (AC) at 3 weeks after RT, total CD3 + T-cells, and CD8 + cytotoxic T-cells distinguished those patients with favorable PFS (≥ 12 months) from all other patients. Discriminant analysis identified B-cells, neutrophil-lymphocyte-ratio (NLR), CD4 + T-helper-cells, and NK-cells as predictors of favorable PFS. High variability in IL-6 plasma concentration of consecutive measurements within 6 months after the end of RT correlated negatively with PFS. Our results suggest that two parameters commonly assessed in clinical routine can be used to predict patient outcome. These are: early increase in CD8 + T-cell lymphocyte count and variability in IL-6 plasma concentration, that are correlated to patients with favorable, respectively, poor outcome after definitive therapy independent of treatment regimen.

Manual expert gating remains common practice for the definition of specific cell populations in the analysis of flow cytometry data. The increasing number of measured parameters per individual cell and high inter-rater variability makes manual gating inconsistent in many scenarios such as multi-center studies. Here, we propose ConvexGating, an AI tool that automatically learns gating strategies in an unbiased, fully data-driven, yet interpretable manner. ConvexGating scales efficiently with increasing parameter space, creating proficient strategies with low-contamination in the extracted population for previously known and so far unknown or ill-defined cell populations. The inferred strategies are independent of parent populations, for instance, plasmacytoid dendritic cells (pDCs) can be fully identified as CD45RA- CD123+. In addition to flow cytometry data, ConvexGating derives gating strategies for cyTOF (Cytometry by Time of Flight) and CITEseq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) data and supports optimal design of marker panels for cell sorting. ### Competing Interest Statement M.B. is currently a full-time employee at Calico Life Sciences, LLC. F.J.T. consults for Immunai Inc., Singularity Bio B.V., CytoReason Ltd, Cellarity Inc., and Curie Bio Operations, LLC, and has an ownership interest in Dermagnostix GmbH and Cellarity Inc. M.S. received funding from Pfizer Inc. for a project related to pneumococcal vaccination. M.S. receives funding from Owkin for a project not related to this research.

Pancreatic ductal adenocarcinoma (PDAC) remains a particularly aggressive disease with few effective treatments. The PDAC tumor immune microenvironment (TIME) is known to be immune suppressive. Oncolytic viruses can increase tumor immunogenicity via immunogenic cell death (ICD). We focused on tumor-selective (vvDD) and cytokine-armed Western-reserve vaccinia viruses (vvDD-IL2 and vvDD-IL15) and infected carcinoma cell lines as well as patient-derived primary PDAC cells. In co-culture experiments, we investigated the cytotoxic response and the activation of human natural killer (NK). Infection and virus replication were assessed by measuring virus encoded YFP. We then analyzed intracellular signaling processes and oncolysis via in-depth proteomic analysis, immunoblotting and TUNEL assay. Following the co-culture of mock or virus infected carcinoma cell lines with allogenic PBMCs or NK cell lines, CD56+ NK cells were analyzed with respect to their activation, cytotoxicity and effector function. Both, dose- and time-dependent release of danger signals following infection were measured. Viruses effectively entered PDAC cells, emitted YFP signals and resulted in concomitant oncolysis. The proteome showed reprogramming of normally active core signaling pathways in PDAC (e.g., MAPK-ERK signaling). Danger-associated molecular patterns were released upon infection and stimulated co-cultured NK cells for enhanced effector cytotoxicity. NK cell subtyping revealed enhanced numbers and activation of a rare CD56dimCD16dim population. Tumor cell killing was primarily triggered via Fas ligands rather than granule release, resulting in marked apoptosis. Overall, the cytokine-armed vaccinia viruses induced NK cell activation and enhanced cytotoxicity toward human PDAC cells in vitro. We could show that cytokine-armed virus targets the carcinoma cells and thus has great potential to modulate the TIME in PDAC.

Background: The immunomodulatory imide drugs (IMiDs) thalidomide, lenalidomide and pomalidomide may exhibit therapeutic efficacy in the prostate. In lower urinary tract symptoms (LUTS), voiding and storage disorders may arise from benign prostate hyperplasia, or overactive bladder. While current therapeutic options target smooth muscle contraction or cell proliferation, side effects are mostly cardiovascular. Therefore, we investigated effects of IMiDs on human detrusor and porcine artery smooth muscle contraction, and growthrelated functions in detrusor smooth muscle cells (HBdSMC). Methods: Cell viability was assessed by CCK8, and apoptosis and cell death by flow cytometry in cultured HBdSMC. Contractions of human detrusor tissues and porcine interlobar and coronary arteries were induced by contractile agonists, or electric field stimulation (EFS) in the presence or absence of an IMID using an organ bath. Proliferation was assessed by EdU assay and colony formation, cytoskeletal organization by phalloidin staining, Results: Depending on tissue type, IMiDs inhibited cholinergic contractions with varying degree, up to 50 %, while non-cholinergic contractions were inhibited up to 80 % and 60 % for U46619 and endothelin-1, respectively, and EFS-induced contractions up to 75 %. IMiDs reduced viable HBdSM cells in a time-dependent manner. Correspondingly, proliferation was reduced, without showing pro-apoptotic effects. In parallel, IMiDs induced cytoskeletal disorganization. Conclusions: IMiDs exhibit regulatory functions in various smooth muscle-rich tissues, and of cell proliferation in the lower urinary tract. This points to a novel drug class effect for IMiDs, in which the molecular mechanisms of action of IMiDs merit further consideration for the application in LUTS.

Background NK cells are the body's first line of defense against cancer, able to recognize and kill tumor cells without having been exposed to the tumor cell previously. Unlike T cells, NK cells do not induce graft-versus-host disease. Therefore, using NK cells in adoptive cell therapy (NK-ACT) holds promise as a true 'off-the-shelf' cellular immunotherapy for cancer, with the potential to circumvent many of the hurdles associated with autologous cell therapies. NK-ACT has demonstrated potential against hematological cancers, but the activity of NK cells in ACT should be enhanced to improve clinical efficacy. Cbl-b, an E3 ubiquitin ligase, is an important gatekeeper, which limits NK cell activation. In NK cells, Cbl-b is activated and stabilized through inhibitory receptor signaling and has been shown to reduce NK cell degranulation and cytotoxicity. Strategies to reduce Cbl-b in NK-ACT products are under active exploration as means to improve the efficacy of NK-ACT. Incorporating RNAi treatment into ex vivo NK cell expansion protocols prior to ACT represents one such strategy. We have developed a new class of stable, self-delivering RNAi compounds (INTASYL™) that incorporate features of RNAi and antisense technology. INTASYL compounds demonstrate potent activity, stability, and are rapidly and efficiently taken up by cells. INTASYL 27457 targeting Cbl-b enhances the cytotoxic activity of expanded human NK cells in vitro. Materials and Methods Primary human CD56+ NK cells were expanded using the ImmunoCult™ NK Cell Expansion Kit. Following the 14-day expansion protocol, cells were seeded directly into 24-well plates containing Cbl-b compound and cultured for 3 days. After the 3-day exposure to INTASYL, compound was washed off and cells were cultured for an additional 3 days. At the end of the culture period, cell viability and expression levels of Cbl-b mRNA were determined. Flow cytometry was performed to explore the impact on various proliferation and activation makers. Cytotoxic capability against the K562 (Chronic Myelogenous Leukemia) cancer cell line was tested in a DELFIA cell cytotoxicity assay. Following co-culture with K562 cells, culture supernatants were analyzed by ELISA for IFN-γ. Results INTASYL treatment resulted in consistent Cbl-b mRNA silencing without negative impact on NK cell viability. Following silencing of Cbl-b markers of NK cell activation and proliferation as well as cytotoxic capability of NK cells against K562 cancer cells were increased. Conclusions We demonstrate the potential of INTASYL self-delivering siRNA targeting Cbl-b to improve NK cell activity for ACT. By treating NK cells with INTASYL targeting the inhibitory protein Cbl-b ex vivo, during NK cell expansion, the proliferation, activation, and anti-tumor response of these cells was enhanced. This may result in a more effective cell therapy for hematological malignancies. M. Maxwell: A. Employment (full or part-time); Significant; Phio Pharmaceuticals. D. Yan: A. Employment (full or part-time); Significant; Phio Pharmaceuticals. B. Rivest: A. Employment (full or part-time); Significant; Phio Pharmaceuticals. J. Cardia: A. Employment (full or part-time); Significant; Phio Pharmaceuticals. E. Noessner: A. Employment (full or part-time); Significant; Helmholtz Zentrum Munich.

Background To explore the impact of dynamic changes of leukocyte subsets during therapy on patient prognosis in inoperable stage III NSCLC, we compared in a prospective explorative single-center study the kinetics of major leukocyte subsets (absolute cell counts, ALC) before, during, and after definitive treatment and correlated it to survival to identify subpopulations associated with maximal patient benefit. Materials and Methods We analyzed peripheral blood of 20 patients with inoperable stage III NSCLC, either treated with radiation therapy (RT), concurrent chemo-radiotherapy (cCRT), or cCRT with additional concurrent (nivolumab) or sequential (durvalumab) immune-checkpoint inhibition therapy. Blood samples were collected at 9 time points including before treatment begin (baseline), at the end of therapy, and during 1 y follow-up after treatment. Analysis included multi-color flow cytometry. Statistical analysis was conducted for leukocyte subpopulations (T cells, B cells, NK cells, neutrophils, thrombocytes), IL-6, progression-free survival (PFS) and overall survival (OS). Results The increase of ALC after the end of thoracic radiotherapy (TRT) until 6 months thereafter was a predictor of PFS. Baseline lymphocyte counts showed no significant correlation to PFS or OS. Early recovery (within three months after TRT) of cell counts in lymphocytes, total CD3+ T cells, and CD8+ cytotoxic T cells were predictors of longer PFS. According to discriminant analysis, B cells, neutrophil-lymphocyte-ratio (NLR), CD4+ T helper cells, and NK cells contributed most to prolonged PFS. Conclusions Our explorative results suggest that early recovery of absolute lymphocyte counts (ALC) of CD8+ T-cells, CD4+ T cells, and NK-cells after (chemo-) radiation-based definitive therapy of inoperable stage III NSCLC is associated with increased PFS. Immune checkpoint therapy showed a trend towards favorable PFS, OS, and fewer local tumor recurrences. T. Hofer: None. A.E. Nieto: None. L. Kaesmann: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; AstraZeneca. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; Amgen. C.J. Pelikan: None. J. Taugner: None. S. Mathur: None. C. Eze: None. C. Belka: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Brainlab, Elekta, ViewRay. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; ViewRay, Bristol Myers Squibb, Roche, Merck & Co, AstraZeneca. F. Consultant/Advisory Board; Modest; ViewRay, Bristol Myers Squibb, Roche, Merck & Co, AstraZeneca, European Society for Radiotherapy and Oncology. F. Manappov: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; AstraZeneca. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; AstraZeneca, Novartis, Roche, Eli Lilly, Elekta, Brainlab. F. Consultant/Advisory Board; Modest; AstraZeneca, Novartis. E. Noessner: None.

Background T cell cytotoxicity plays a crucial role in defending against cancer. It is traditionally assessed by endpoint assays with non-microscopic appliance, thus lacking temporal and visual insights into middle time ranges that live-cell imaging offers. However, conventional live-cell imaging falls short in data scale due to limited replicates per condition. Combining micro-structured slides with live-cell imaging addresses these issues. Multiple cell adhesion patterns enable independent progression of killing processes, providing multiplicates of T cell-mediated killing events in one experiment. Handling the high data volume manually can be cumbersome and biased, calling for an objective means of data processing. We were analyzing two cytotoxic T cells with different avidities as determined by IFN-γ release, but failed to clearly differentiate killing capacities using chromium release assay. Therefore, we asked if micro-pattern and AI-assisted analysis could resolve differences in killing. Materials and Methods Human primary T cells were transduced with T cell receptors of different avidities for the same peptide/MHC complex (T58, D115). D115-T cells were additionally engineered to express chimeric costimulatory switch proteins (CSPs). These CSPs were created by linking the extracellular domain of PD-1 with an intracellular costimulatory domain of CD28 or 4-1BB. Functional T cell avidities were defined by IFN-γ release after co-culture with SkMel23 melanoma cells. Live-cell imaging used micro-patterned µ-Slides 8-Well (ibidi). T cells and tumor cells were used label-free to avoid potential effects on killing processes. Data analysis employed Cellpose, an AI-powered cellular segmentation tool with human-in-the-loop features. Manual quality control and correction, if needed, adopted the interactive tool we built with Python and Jupyter Notebook. R was used to interpret the formatted data. Results We acquired time series of tumor cell/T cell co-cultures on micro-patterned µ-Slides 8-Well. Cellpose was trained to precisely enclose label-free tumor cell patches formed on the micro-structures. The segmentation was integrated into an automated workflow, whereby the AI automation recognized the outline of tumor cell patches on the micro-structures in sequential time frames and was able to follow each patch over time even if its confluency changed. Applying the live-cell imaging set-up with micro-structured slides together with AI-powered analysis, we were able to resolve different dynamics of confluency changes for high and low avidity T cells indicating different killing behaviors. The automation is now applied to evaluate killing efficacies of T cells with CSP expression and T cells exposed to various components of the tumor microenvironment. Conclusions Micro-patterns provide standardized cell patches and, hence, high multiplicity within one experiment for statistical robustness. Pattern confluency is reliably measured with trained AI software allowing for processing of high-throughput live-cell imaging data which is essential for selecting T cell variants which best perform in given conditions like the complex tumor microenvironment. Support JC is funded by China Scholarship Council (No. 202306380076), AF, EN, MS and DR by ZIM/AppMic, and AH by Phio Pharmaceuticals. J. Cao: None. W. Xu: None. A.J. Fischbeck: None. A.S. Herbstritt: C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Significant; Phio Pharmaceuticals. S. Prins: None. J. Rädler: None. E. Noessner: None. M. Seiwald: A. Employment (full or part-time); Significant; ibidi GmbH. D. Rüdiger: A. Employment (full or part-time); Significant; ibidi GmbH.

You have accessJournal of UrologyCME1 Apr 2023PD23-07 IMMUNO-MODULATORY IMIDE DRUGS (IMIDS) INHIBIT HUMAN BLADDER SMOOTH MUSCLE CONTRACTION: A NOVEL DRUG CLASS IN LUTS? Alexander Tamalunas, Amin Wendt, Florian Springer, Victor Vigodski, Ru Huang, Ruixiao Wang, Yuhan Liu, Beata Rutz, Anna Ciotkowska, Giuseppe Magistro, Christian Stief, and Martin Hennenberg Alexander TamalunasAlexander Tamalunas More articles by this author , Amin WendtAmin Wendt More articles by this author , Florian SpringerFlorian Springer More articles by this author , Victor VigodskiVictor Vigodski More articles by this author , Ru HuangRu Huang More articles by this author , Ruixiao WangRuixiao Wang More articles by this author , Yuhan LiuYuhan Liu More articles by this author , Beata RutzBeata Rutz More articles by this author , Anna CiotkowskaAnna Ciotkowska More articles by this author , Giuseppe MagistroGiuseppe Magistro More articles by this author , Christian StiefChristian Stief More articles by this author , and Martin HennenbergMartin Hennenberg More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003296.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Medical treatment in lower urinary tract symptoms (LUTS) includes reduction of prostate and detrusor smooth muscle tone. Due to an unfavourable balance between side effects and limited efficacy, current medications cause high rates of patient non-compliance and treatment discontinuation. Recently, we could show that IMiDs (thalidomide, lenalidomide and pomalidomide) inhibit prostate smooth muscle contraction, modulate cytoskeletal actin organization, and reduce prostate stromal cell growth, without causing cytotoxic side effects. Here, we addressed effects of IMiDs on cholinergic and non-cholinergic contractions of human detrusor smooth muscle. METHODS: Detrusor wall tissues were obtained from patients undergoing radical cystectomy for bladder cancer (n=83 patients). Contractility of detrusor strips was then assessed in an organ bath. RESULTS: IMiDs ([1] thalidomide 100µM, [2] lenalidomide 20µM, and [3] pomalidomide 5µM) inhibited cholinergic contractions at a varying degree, by 27-39% and 30-43% for concentrations of 0.1-1000µM carbachol and methacholine, respectively. Non-cholinergic contractions were inhibited by 44-50% and 37-53% for thromboxane A2 analogue U46619, and endothelin-1, respectively. While thalidomide and lenalidomide had no or only marginal effect on EFS-induced neurogenic contractions, pomalidomide inhibited EFS-induced contractions by 35%. CONCLUSIONS: Together with our previous data on prostate smooth muscle contraction, the observed varying susceptibility to cholinergic, non-cholinergic, and neurogenic contractions suggests specificity and differences in IMiDs on smooth muscle contractility in the lower urinary tract, and a possible novel drug class in LUTS treatment. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e673 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alexander Tamalunas More articles by this author Amin Wendt More articles by this author Florian Springer More articles by this author Victor Vigodski More articles by this author Ru Huang More articles by this author Ruixiao Wang More articles by this author Yuhan Liu More articles by this author Beata Rutz More articles by this author Anna Ciotkowska More articles by this author Giuseppe Magistro More articles by this author Christian Stief More articles by this author Martin Hennenberg More articles by this author Expand All Advertisement PDF downloadLoading ...

Background Adoptive T cell therapy (ATT) has shown efficacy in the treatment of hematologic malignancies. However, in solid cancer, response rates are currently unsatisfactorily low. One of the major challenges in the treatment of solid tumors is to overcome the poor lifespan and the loss of function of tumor-reactive T cells in the tumor microenvironment (TME). We aim to overcome these hurdles by engineering tumor-reactive T cells with chimeric costimulatory switch proteins (CSPs) which should prevent PD-1/L1-mediated inhibition and simultaneous turning it into activation of costimulatory pathways leading to functional enhancement and prolonged survival. Materials and Methods Two CSPs were generated combining the extracellular domain of PD-1 with an intracellular costimulatory domain of either CD28 or 4-1BB. T cells without CSP are controls. Primary human tumor-reactive CD8 T cells were engineered to express T cell receptors (TCRs) with HLA-A2 restricted recognition of either a tyrosinase or a renal cell carcinoma (RCC) peptide. CSP-engineered TCR-T cells and controls were in-depth characterized regarding cytokine release and cytotoxicity, phenotype, differentiation and metabolic state in vitro after co-culture (2D/3D-matrigel) in TME-adapted conditions (using melanoma or renal cell carcinoma (RCC) cell lines) and in vivo using an orthotopic human RCC mouse model. Blood, tumors and organs were analyzed for the presence and phenotype of T cells by flow cytometry. Results In vitro, CSP-engineered TCR-T cells showed enhanced cytokine release and cytotoxicity. CSPs enabled maintenance of function during repeated tumor challenge and nutrient-restricted conditions. Flow cytometry identified higher Ki67, IRF4 and BATF as well as markers of metabolic fitness in CSP-TCR-T cells compared to control T cells following co-culture with PD-L1 positive tumor cells. Induced changes were dependent on tumor cells expressing PD-L1 in the context of peptide/MHC ligands. In vivo, using an orthotopic implant of RCC tumor cells, ATT with TCR-T cells reduced tumor volume compared to the no T cell group, and only residual tumor cells were detected after histological examination in the CSP-TCR-T cell groups. Plasma IFNγ levels at day 3 after ATT were higher for CSP-TCR-T cells compared to control T cells. Conclusions We collected essential functional data using in vitro and in vivo models describing effects of PD-1-based CSPs on TCR-T cell phenotype and functional activity. We observed higher efficacy of PD-1-CSP-T cells in controlling orthotopic RCC xenografts, which is to our knowledge the first report of PD-1-CSP effects using non-affinity-enhanced TCR (isolated from human tumor-infiltrating T cells) and unmodified cancer cells with endogenous (not engineered) antigen/MHC and PD-L1. The results suggest that PD-1-CSPs can empower tumor antigen-reactive TCR-T cells for beneficial application in solid tumor therapy paving the way for in human clinical trials using TCR-T cells armored with CSPs. Funded by Deutsche Krebshilfe, and ZIM/AppMic. E. Noessner: None. C. Aman: None. A.J. Fischbeck: None. K. Junker: None. J. Linxweiler: None.

Natural killer (NK) cells are frontline defenders against cancer and are capable of recognizing and eliminating tumor cells without prior sensitization or antigen presentation. Due to their unique HLA mismatch tolerance, they are ideal for adoptive cell therapy (ACT) because of their ability to minimize graft-versus-host-disease risk. The therapeutic efficacy of NK cells is limited in part by inhibitory immune checkpoint receptors, which are upregulated upon interaction with cancer cells and the tumor microenvironment. Overexpression of inhibitory receptors reduces NK cell-mediated cytotoxicity by impairing the ability of NK cells to secrete effector cytokines and cytotoxic granules. T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), a well-known checkpoint receptor involved in T-cell exhaustion, has recently been implicated in the exhaustion of NK cells. Overcoming TIGIT-mediated inhibition of NK cells may allow for a more potent antitumor response following ACT. Here, we describe a novel approach to TIGIT inhibition using self-delivering RNAi compounds (INTASYL™) that incorporates the features of RNAi and antisense technology. INTASYL compounds demonstrate potent activity and stability, are rapidly and efficiently taken up by cells, and can be easily incorporated into cell product manufacturing. INTASYL PH-804, which targets TIGIT, suppresses TIGIT mRNA and protein expression in NK cells, resulting in increased cytotoxic capacity and enhanced tumor cell killing in vitro. Delivering PH-804 to NK cells before ACT has emerged as a promising strategy to counter TIGIT inhibition, thereby improving the antitumor response. This approach offers the potential for more potent off-the-shelf products for adoptive cell therapy, particularly for hematological malignancies.

ABSTRACT Background Epstein-Barr virus-associated Infectious Mononucleosis (EBV-IM) is a common disease following primary EBV infection in children and adolescents. While EBV-IM is mostly self-limiting, symptoms like fatigue may persist over several months or even result in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). This large clinical observational study aimed at identifying risk factors for protracted courses of EBV-IM in young people. Methods A cohort of N=200 children, adolescents, and young adults with acute primary EBV infection was recruited from hospitals and private practices. Data on the patients’ medical history as well as clinical and laboratory parameters were collected at a baseline visit (V1) within four weeks after symptom onset (T onset ) and at two follow-up visits (V2 and V3) one and six months after T onset . Risk factors for protracted symptoms at V3 were modeled using multivariable logistic regressions. Results Protracted symptoms were observed in 55/183 (30.1%) and protracted fatigue in 34/181 (18.8%) patients at V3. A medical history indicating an increased susceptibility to infectious diseases as well as distinct severe IM symptoms, e.g. severe gastrointestinal symptoms, were significantly associated with protracted disease [OR: 2.31; P=0.011 and OR: 3.42; P=0.027] and with chronic fatigue [OR: 2.98; P=0.006 and OR: 3.54; P=0.034], respectively. Occurrence of twelve or more clinical and laboratory parameters until and including V1 discriminated between fatigue and no fatigue at V3 [OR 2.43, P=0.033]. Conclusion A clinical history of immune dysregulation as well as distinct severe IM symptoms might predict protracted post-viral disease and thus help in the identification of young patients at risk. Highlights Severe gastrointestinal symptoms are associated with protracted course of Epstein-Barr virus-associated Infectious Mononucleosis (EBV-IM). Signs of immune dysregulation prior to EBV-IM can indicate an increased risk of protracted symptoms. Greater number of initial symptoms helps to identify patients developing postviral fatigue.

BACKGROUND:Virus infections drive COPD exacerbations and progression. Antiviral immunity centres on the activation of virus-specific CD8+ T-cells by viral epitopes presented on major histocompatibility complex (MHC) class I molecules of infected cells. These epitopes are generated by the immunoproteasome, a specialised intracellular protein degradation machine, which is induced by antiviral cytokines in infected cells.METHODS:We analysed the effects of cigarette smoke on cytokine- and virus-mediated induction of the immunoproteasome in vitro, ex vivo and in vivo using RNA and Western blot analyses. CD8+ T-cell activation was determined in co-culture assays with cigarette smoke-exposed influenza A virus (IAV)-infected cells. Mass-spectrometry-based analysis of MHC class I-bound peptides uncovered the effects of cigarette smoke on inflammatory antigen presentation in lung cells. IAV-specific CD8+ T-cell numbers were determined in patients' peripheral blood using tetramer technology.RESULTS:Cigarette smoke impaired the induction of the immunoproteasome by cytokine signalling and viral infection in lung cells in vitro, ex vivo and in vivo. In addition, cigarette smoke altered the peptide repertoire of antigens presented on MHC class I molecules under inflammatory conditions. Importantly, MHC class I-mediated activation of IAV-specific CD8+ T-cells was dampened by cigarette smoke. COPD patients exhibited reduced numbers of circulating IAV-specific CD8+ T-cells compared to healthy controls and asthmatics.CONCLUSION:Our data indicate that cigarette smoke interferes with MHC class I antigen generation and presentation and thereby contributes to impaired activation of CD8+ T-cells upon virus infection. This adds important mechanistic insight on how cigarette smoke mediates increased susceptibility of smokers and COPD patients to viral infections.