Preclinical Development of CAR T Cells with Antigen-Inducible IL18 Enforcement to Treat GD2-Positive Solid Cancers.

PURPOSE: Cytokine-engineering of chimeric antigen receptor-redirected T cells (CAR T cells) is a promising principle to overcome the limited activity of canonical CAR T cells against solid cancers. EXPERIMENTAL DESIGN: We developed an investigational medicinal product, GD2IL18CART, consisting of CAR T cells directed against ganglioside GD2 with CAR-inducible IL18 to enhance their activation response and cytolytic effector functions in the tumor microenvironment. To allow stratification of patients according to tumor GD2 expression, we established and validated immunofluorescence detection of GD2 on paraffin-embedded tumor tissues. RESULTS: Lentiviral all-in-one vector engineering of human T cells with the GD2-specific CAR with and without inducible IL18 resulted in cell products with comparable proportions of CAR-expressing central memory T cells. Production of IL18 strictly depends on GD2 antigen engagement. GD2IL18CART respond to interaction with GD2-positive tumor cells with higher IFNγ and TNFα cytokine release and more effective target cytolysis compared with CAR T cells without inducible IL18. GD2IL18CART further have superior in vivo antitumor activity, with eradication of GD2-positive tumor xenografts. Finally, we established GMP-compliant manufacturing of GD2IL18CART and found it to be feasible and efficient at clinical scale. CONCLUSIONS: These results pave the way for clinical investigation of GD2IL18CART in pediatric and adult patients with neuroblastoma and other GD2-positive cancers (EU CT 2022-501725-21-00).

Comparative Gene Signature of Nociceptors Innervating Mouse Molar Teeth, Cranial Meninges, and Cornea.

BACKGROUND: The trigeminal ganglion (TG) collects afferent sensory information from various tissues. Recent large-scale RNA sequencing of neurons of the TG and dorsal root ganglion has revealed a variety of functionally distinct neuronal subpopulations, but organ-specific information is lacking. METHODS: To link transcriptomic and tissue-specific information, we labeled small-diameter neurons of 3 specific subpopulations of the TG by local application of lipophilic carbocyanine dyes to their innervation site in the dental pulp, cornea, and meninges (dura mater). We then collected mRNA-sequencing data from fluorescent neurons. Differentially expressed genes (DEGs) were analyzed and subjected to downstream gene set enrichment analysis (GSEA), and ion channel profiling was performed. RESULTS: A total of 10,903 genes were mapped to the mouse genome (>500 reads). DEG analysis revealed 18 and 81 genes with differential expression (log2 fold change > 2, Padj < .05) in primary afferent neurons innervating the dental pulp (dental primary afferent neurons [DPAN]) compared to those innervating the meninges (meningeal primary afferent neurons [MPAN]) and the cornea (corneal primary afferent neurons [CPAN]). We found 250 and 292 genes differentially expressed in MPAN as compared to DPAN and to CPAN, and 21 and 12 in CPAN as compared to DPAN and MPAN. Scn2b had the highest log2 fold change when comparing DPAN versus MPAN and Mmp12 was the most prominent DEG when comparing DPAN versus CPAN and, CPAN versus MPAN. GSEA revealed genes of the immune and mitochondrial oxidative phosphorylation system for the DPAN versus MPAN comparison, cilium- and ribosome-related genes for the CPAN versus DPAN comparison, and respirasome, immune cell- and ribosome-related gene sets for the CPAN versus MPAN comparison. DEG analysis for ion channels revealed no significant differences between the neurons set except for the sodium voltage-gated channel beta subunit 2, Scn2b. However, in each tissue a few ion channels turned up with robust number of reads. In DPAN, these were Cacna1b, Trpv2, Cnga4, Hcn1, and Hcn3, in CPAN Trpa1, Trpv1, Cacna1a, and Kcnk13 and in MPAN Trpv2 and Scn11a. CONCLUSIONS: Our study uncovers previously unknown differences in gene expression between sensory neuron subpopulations from the dental pulp, cornea, and dura mater and provides the basis for functional studies, including the investigation of ion channel function and their suitability as targets for tissue-specific analgesia.

TRPM8-dependent shaking in mammals and birds.

Removing water from wet fur or feathers is important for thermoregulation in warm-blooded animals. The "wet dog shake" (WDS) behavior has been largely characterized in mammals but to a much lesser extent in birds. Although it is known that TRPM8 is the main molecular transducer of low temperature in mammals, it is not clear if wetness-induced shaking in furred and feathered animals is dependent on TRPM8. Here, we show that a novel TRPM8 agonist induces WDS in rodents and, importantly, in birds, similar to the shaking behavior evoked by water-spraying. Furthermore, the WDS onset depends on TRPM8, as we show in water-sprayed mice. Overall, our results provide multiple evidence for a TRPM8 dependence of WDS behaviors in all tested species. These suggest that a convergent evolution selected similar shaking behaviors to expel water from fur and feathers, with TRPM8 being involved in wetness sensing in both mammals and birds.

Interprofessional Therapeutic Drug Monitoring of Carbapenems Improves ICU Care and Guideline Adherence in Acute-on-Chronic Liver Failure.

(1) Background: Acute-on-chronic liver failure (ACLF) is a severe, rapidly progressing disease in patients with liver cirrhosis. Meropenem is crucial for treating severe infections. Therapeutic drug monitoring (TDM) offers an effective means to control drug dosages, especially vital for bactericidal antibiotics like meropenem. We aimed to assess the outcomes of implementing TDM for meropenem using an innovative interprofessional approach in ACLF patients on a medical intensive care unit (ICU). (2) Methods: The retrospective study was conducted on a medical ICU. The outcomes of an interprofessional approach comprising physicians, hospital pharmacists, and staff nurses to TDM for meropenem in critically ill patients with ACLF were examined in 25 patients. Meropenem was administered continuously via an infusion pump after the application of an initial loading dose. TDM was performed weekly using high-performance liquid chromatography (HPLC). Meropenem serum levels, implementation of the recommendations of the interprofessional team, and meropenem consumption were analyzed. (3) Results: Initial TDM for meropenem showed a mean meropenem serum concentration of 20.9 ± 9.6 mg/L in the 25 analyzed patients. Of note, in the initial TDM, only 16.0% of the patients had meropenem serum concentrations within the respective target range, while 84.0% exceeded this range. Follow-up TDM showed serum concentrations of 15.2 ± 5.7 mg/L (9.0-24.6) in Week 2 and 11.9 ± 2.3 mg/L (10.2-13.5) in Week 3. In Week 2, 41.7% of the patients had meropenem serum concentrations that were within the respective target range, while 58.3% of the patients were above this range. In Week 3, 50% of the analyzed serum concentrations of meropenem were within the targeted range, and 50% were above the range. In total, 100% of the advice given by the interprofessional team regarding meropenem dosing or a change in antibiotic therapy was implemented. During the intervention period, the meropenem application density was 37.9 recommended daily doses (RDD)/100 patient days (PD), compared to 42.1 RDD/100 PD in the control period, representing a 10.0% decrease. (4) Conclusions: Our interprofessional approach to TDM significantly reduced meropenem dosing, with all the team's recommendations being implemented. This method not only improved patient safety but also considerably decreased the application density of meropenem.

CAR-Ts redirected against the Thomsen-Friedenreich antigen CD176 mediate specific elimination of malignant cells from leukemia and solid tumors.

Introduction: Chimeric antigen receptor-engineered T cells (CAR-Ts) are investigated in various clinical trials for the treatment of cancer entities beyond hematologic malignancies. A major hurdle is the identification of a target antigen with high expression on the tumor but no expression on healthy cells, since "on-target/off-tumor" cytotoxicity is usually intolerable. Approximately 90% of carcinomas and leukemias are positive for the Thomsen-Friedenreich carbohydrate antigen CD176, which is associated with tumor progression, metastasis and therapy resistance. In contrast, CD176 is not accessible for ligand binding on healthy cells due to prolongation by carbohydrate chains or sialylation. Thus, no "on-target/off-tumor" cytotoxicity and low probability of antigen escape is expected for corresponding CD176-CAR-Ts. Methods: Using the anti-CD176 monoclonal antibody (mAb) Nemod-TF2, the presence of CD176 was evaluated on multiple healthy or cancerous tissues and cells. To target CD176, we generated two different 2nd generation CD176-CAR constructs differing in spacer length. Their specificity for CD176 was tested in reporter cells as well as primary CD8+ T cells upon co-cultivation with CD176+ tumor cell lines as models for CD176+ blood and solid cancer entities, as well as after unmasking CD176 on healthy cells by vibrio cholerae neuraminidase (VCN) treatment. Following that, both CD176-CARs were thoroughly examined for their ability to initiate target-specific T-cell signaling and activation, cytokine release, as well as cytotoxicity. Results: Specific expression of CD176 was detected on primary tumor tissues as well as on cell lines from corresponding blood and solid cancer entities. CD176-CARs mediated T-cell signaling (NF-κB activation) and T-cell activation (CD69, CD137 expression) upon recognition of CD176+ cancer cell lines and unmasked CD176, whereby a short spacer enabled superior target recognition. Importantly, they also released effector molecules (e.g. interferon-γ, granzyme B and perforin), mediated cytotoxicity against CD176+ cancer cells, and maintained functionality upon repetitive antigen stimulation. Here, CD176L-CAR-Ts exhibited slightly higher proliferation and mediator-release capacities. Since both CD176-CAR-Ts did not react towards CD176- control cells, their response proved to be target-specific. Discussion: Genetically engineered CD176-CAR-Ts specifically recognize CD176 which is widely expressed on cancer cells. Since CD176 is masked on most healthy cells, this antigen and the corresponding CAR-Ts represent a promising approach for the treatment of various blood and solid cancers while avoiding "on-target/off-tumor" cytotoxicity.

Planetary health as a main topic for the qualification in digital teaching - a project report.

Aim: To do justice to the need for planetary health in medical education, these are the aims of the online elective course "Planetary Health in Medical Education" (ME elective):1. Enable students to plan and realize their own course sessions on planetary health;2. Encourage communication among university medical faculties regarding planetary health in medical education;3. Reinforce competency in digital teaching and amplify the expert role as multiplicator among students pursuing a Master's degree in Medicinal Education (MME). Method: The development of the ME elective followed Kern's six-step approach to curriculum development by means of cooperation between the German Medical Students' Association (Bundesvertretung der Medizinstudierenden in Deutschland, abbreviated as bvmd), and the MME study program. Based on general and specific needs analyses, core learning objectives regarding planetary health, medical education and digital education were identified in the National Catalogue of Learning Objectives in Undergraduate Medical Education (NKLM) and the MME study program and relevant teaching methods were selected. Results: The ME elective, consisting of two contact hours per week per semester, was established at 13 medical schools as a four-phase course:1. Introduction to medical education using examples from planetary health;2. Lesson planning on a topic in planetary health under the supervision of MME students;3. Course sessions held by the undergraduate students; and4. Networking with the MME study program through participation in digital courses on planetary health and the pilot OSCE on planetary health.A total of 24 students attended the pilot in the 2022 summer semester. Conclusion: The topic of planetary health combines interests that span many subjects and semester levels. As a collaborative, interdisciplinary and interprofessional subject, it lends itself to training students in a trans-institutional elective course to become multiplicators.

CAR-NK Cells Targeting HER1 (EGFR) Show Efficient Anti-Tumor Activity against Head and Neck Squamous Cell Carcinoma (HNSCC).

(1) Background: HNSCC is a highly heterogeneous and relapse-prone form of cancer. We aimed to expand the immunological tool kit against HNSCC by conducting a functional screen to generate chimeric antigen receptor (CAR)-NK-92 cells that target HER1/epidermal growth factor receptor (EGFR). (2) Methods: Selected CAR-NK-92 cell candidates were tested for enhanced reduction of target cells, CD107a expression and IFNγ secretion in different co-culture models. For representative HNSCC models, patient-derived primary HNSCC (pHNSCC) cell lines were generated by employing an EpCAM-sorting approach to eliminate the high percentage of non-malignant cells found. (3) Results: 2D and 3D spheroid co-culture experiments showed that anti-HER1 CAR-NK-92 cells effectively eliminated SCC cell lines and primary HNSCC (pHNSCC) cells. Co-culture of tumor models with anti-HER1 CAR-NK-92 cells led to enhanced degranulation and IFNγ secretion of NK-92 cells and apoptosis of target cells. Furthermore, remaining pHNSCC cells showed upregulated expression of putative cancer stem cell marker CD44v6. (4) Conclusions: These results highlight the promising potential of CAR-NK cell therapy in HNSCC and the likely necessity to target multiple tumor-associated antigens to reduce currently high relapse rates.

Generalized resistance to pruritogen-induced scratching in the C3H/HeJ strain.

Previously the effect of the pruritogens, such as histamine and chloroquine, was tested in 11 inbred mouse strains, and this study aimed to identify resistant and sensitive strains, consistent with the observation that underlies the large variability in human populations. In the present study, we used the low responder C3H/HeJ (C3H) and the more sensitive C57BL/6J (C57) strain to find out if resistance and sensitivity to develop pruritus is restricted to only histamine and chloroquine or extends to other known pruritogens as well. We tested five additional commonly known pruritogens. We established dose-response relationships by injecting four concentrations of the pruritogens in the range of 0.3, 1, 3, and ten-fold in the nuchal fold. Then we assessed the scratching behavior for 30 min after injection with an automated custom-designed device based on the bilateral implantation of mini-magnets in the hind paws and on single cages placed within a magnetic coil. We found that the resistance to pruritogens is a general phenotype of the C3H strain and extends to all pruritogens tested, including not only histamine and chloroquine, but also endothelin, trypsin, 5-HT (serotonin), the short peptide SLIGRL, and Lysophosphatidic acid (LPA). C57 was more sensitive to all pruritogens and, in contrast to C3H, dose-response relationships were evident for some of the pruritogens. In general, comparable peak scratch responses were observed for the 0.3-fold concentrations of the pruritogens in C57 whereas C3H required at least the ten-fold concentration and still displayed only between 5 and 33% of the scratch responses observed in C57 for the respective pruritogen. The general resistance to pruritogens and the low level of scratching behavior found in the C3H strain is an interesting trait and represents a model for the study of the heritability of itch. It is accompanied in C3H with a higher sensitivity in assays of nociception.

MCU controls melanoma progression through a redox-controlled phenotype switch.

Melanoma is the deadliest of skin cancers and has a high tendency to metastasize to distant organs. Calcium and metabolic signals contribute to melanoma invasiveness; however, the underlying molecular details are elusive. The MCU complex is a major route for calcium into the mitochondrial matrix but whether MCU affects melanoma pathobiology was not understood. Here, we show that MCUA expression correlates with melanoma patient survival and is decreased in BRAF kinase inhibitor-resistant melanomas. Knockdown (KD) of MCUA suppresses melanoma cell growth and stimulates migration and invasion. In melanoma xenografts, MCUA_KD reduces tumor volumes but promotes lung metastases. Proteomic analyses and protein microarrays identify pathways that link MCUA and melanoma cell phenotype and suggest a major role for redox regulation. Antioxidants enhance melanoma cell migration, while prooxidants diminish the MCUA_KD -induced invasive phenotype. Furthermore, MCUA_KD increases melanoma cell resistance to immunotherapies and ferroptosis. Collectively, we demonstrate that MCUA controls melanoma aggressive behavior and therapeutic sensitivity. Manipulations of mitochondrial calcium and redox homeostasis, in combination with current therapies, should be considered in treating advanced melanoma.

GMP-Compliant Manufacturing of TRUCKs: CAR T Cells targeting GD2 and Releasing Inducible IL-18.

Chimeric antigen receptor (CAR)-engineered T cells can be highly effective in the treatment of hematological malignancies, but mostly fail in the treatment of solid tumors. Thus, approaches using 4th advanced CAR T cells secreting immunomodulatory cytokines upon CAR signaling, known as TRUCKs ("T cells redirected for universal cytokine-mediated killing"), are currently under investigation. Based on our previous development and validation of automated and closed processing for GMP-compliant manufacturing of CAR T cells, we here present the proof of feasibility for translation of this method to TRUCKs. We generated IL-18-secreting TRUCKs targeting the tumor antigen GD2 using the CliniMACS Prodigy® system using a recently described "all-in-one" lentiviral vector combining constitutive anti-GD2 CAR expression and inducible IL-18. Starting with 0.84 x 108 and 0.91 x 108 T cells after enrichment of CD4+ and CD8+ we reached 68.3-fold and 71.4-fold T cell expansion rates, respectively, in two independent runs. Transduction efficiencies of 77.7% and 55.1% was obtained, and yields of 4.5 x 109 and 3.6 x 109 engineered T cells from the two donors, respectively, within 12 days. Preclinical characterization demonstrated antigen-specific GD2-CAR mediated activation after co-cultivation with GD2-expressing target cells. The functional capacities of the clinical-scale manufactured TRUCKs were similar to TRUCKs generated in laboratory-scale and were not impeded by cryopreservation. IL-18 TRUCKs were activated in an antigen-specific manner by co-cultivation with GD2-expressing target cells indicated by an increased expression of activation markers (e.g. CD25, CD69) on both CD4+ and CD8+ T cells and an enhanced release of pro-inflammatory cytokines and cytolytic mediators (e.g. IL-2, granzyme B, IFN-γ, perforin, TNF-α). Manufactured TRUCKs showed a specific cytotoxicity towards GD2-expressing target cells indicated by lactate dehydrogenase (LDH) release, a decrease of target cell numbers, microscopic detection of cytotoxic clusters and detachment of target cells in real-time impedance measurements (xCELLigence). Following antigen-specific CAR activation of TRUCKs, CAR-triggered release IL-18 was induced, and the cytokine was biologically active, as demonstrated in migration assays revealing specific attraction of monocytes and NK cells by supernatants of TRUCKs co-cultured with GD2-expressing target cells. In conclusion, GMP-compliant manufacturing of TRUCKs is feasible and delivers high quality T cell products.

Generation of an NFκB-Driven Alpharetroviral "All-in-One" Vector Construct as a Potent Tool for CAR NK Cell Therapy.

Immune cell therapeutics are increasingly applied in oncology. Especially chimeric antigen receptor (CAR) T cells are successfully used to treat several B cell malignancies. Efforts to engineer CAR T cells for improved activity against solid tumors include co-delivery of pro-inflammatory cytokines in addition to CARs, via either constitutive cytokine expression or inducible cytokine expression triggered by CAR recognition of its target antigen-so-called "T cells redirected for universal cytokine-mediated killing" (TRUCKs) or fourth-generation CARs. Here, we tested the hypothesis that TRUCK principles could be expanded to improve anticancer functions of NK cells. A comparison of the functionality of inducible promoters responsive to NFAT or NFκB in NK cells showed that, in contrast to T cells, the inclusion of NFκB-responsive elements within the inducible promoter construct was essential for CAR-inducible expression of the transgene. We demonstrated that GD2CAR-specific activation induced a tight NFκB-promoter-driven cytokine release in NK-92 and primary NK cells together with an enhanced cytotoxic capacity against GD2+ target cells, also shown by increased secretion of cytolytic cytokines. The data demonstrate biologically relevant differences between T and NK cells that are important when clinically translating the TRUCK concept to NK cells for the treatment of solid malignancies.

NK Cell-Mediated Eradication of Ovarian Cancer Cells with a Novel Chimeric Antigen Receptor Directed against CD44.

Ovarian cancer is the most common cause of gynecological cancer-related death in the developed world. Disease recurrence and chemoresistance are major causes of poor survival rates in ovarian cancer patients. Ovarian cancer stem cells (CSCs) were shown to represent a source of tumor recurrence owing to the high resistance to chemotherapy and enhanced tumorigenicity. Chimeric antigen receptor (CAR)-based adoptive immunotherapy represents a promising strategy to reduce the risk for recurrent disease. In this study, we developed a codon-optimized third-generation CAR to specifically target CD44, a marker widely expressed on ovarian cancer cells and associated with CSC-like properties and intraperitoneal tumor spread. We equipped NK-92 cells with the anti-CD44 CAR (CD44NK) and an anti-CD19 control CAR (CD19NK) using lentiviral SIN vectors. Compared to CD19NK and untransduced NK-92 cells, CD44NK showed potent and specific cytotoxic activity against CD44-positive ovarian cancer cell lines (SKOV3 and OVCAR3) and primary ovarian cancer cells harvested from ascites. In contrast, CD44NK had less cytotoxic activity against CD44-negative A2780 cells. Specific activation of engineered NK cells was also demonstrated by interferon-γ (IFNγ) secretion assays. Furthermore, CD44NK cells still demonstrated cytotoxic activity under cisplatin treatment. Most importantly, the simultaneous treatment with CD44NK and cisplatin showed higher anti-tumor activity than sequential treatment.

Improved Activity against Acute Myeloid Leukemia with Chimeric Antigen Receptor (CAR)-NK-92 Cells Designed to Target CD123.

Anti-cancer activity can be improved by engineering immune cells to express chimeric antigen receptors (CARs) that recognize tumor-associated antigens. Retroviral vector gene transfer strategies allow stable and durable transgene expression. Here, we used alpharetroviral vectors to modify NK-92 cells, a natural killer cell line, with a third-generation CAR designed to target the IL-3 receptor subunit alpha (CD123), which is strongly expressed on the surface of acute myeloid leukemia (AML) cells. Alpharetroviral vectors also contained a transgene cassette to allow constitutive expression of human IL-15 for increased NK cell persistence in vivo. The anti-AML activity of CAR-NK-92 cells was tested via in vitro cytotoxicity assays with the CD123+ AML cell line KG-1a and in vivo in a patient-derived xenotransplantation CD123+ AML model. Unmodified NK-92 cells or NK-92 cells modified with a truncated version of the CAR that lacked the signaling domain served as controls. Alpharetroviral vector-modified NK-92 cells stably expressed the transgenes and secreted IL-15. Anti-CD123-CAR-NK-92 cells exhibited enhanced anti-AML activity in vitro and in vivo as compared to control NK-92 cells. Our data (1) shows the importance of IL-15 expression for in vivo persistence of NK-92 cells, (2) supports continued investigation of anti-CD123-CAR-NK cells to target AML, and (3) points towards potential strategies to further improve CAR-NK anti-AML activity.

Odontoblast TRPC5 channels signal cold pain in teeth.

Teeth are composed of many tissues, covered by an inflexible and obdurate enamel. Unlike most other tissues, teeth become extremely cold sensitive when inflamed. The mechanisms of this cold sensation are not understood. Here, we clarify the molecular and cellular components of the dental cold sensing system and show that sensory transduction of cold stimuli in teeth requires odontoblasts. TRPC5 is a cold sensor in healthy teeth and, with TRPA1, is sufficient for cold sensing. The odontoblast appears as the direct site of TRPC5 cold transduction and provides a mechanism for prolonged cold sensing via TRPC5's relative sensitivity to intracellular calcium and lack of desensitization. Our data provide concrete functional evidence that equipping odontoblasts with the cold-sensor TRPC5 expands traditional odontoblast functions and renders it a previously unknown integral cellular component of the dental cold sensing system.

CAR-T cells and TRUCKs that recognize an EBNA-3C-derived epitope presented on HLA-B*35 control Epstein-Barr virus-associated lymphoproliferation.

BACKGROUND: Immunosuppressive therapy or T-cell depletion in transplant patients can cause uncontrolled growth of Epstein-Barr virus (EBV)-infected B cells resulting in post-transplant lymphoproliferative disease (PTLD). Current treatment options do not distinguish between healthy and malignant B cells and are thereby often limited by severe side effects in the already immunocompromised patients. To specifically target EBV-infected B cells, we developed a novel peptide-selective chimeric antigen receptor (CAR) based on the monoclonal antibody TÜ165 which recognizes an Epstein-Barr nuclear antigen (EBNA)-3C-derived peptide in HLA-B*35 context in a T-cell receptor (TCR)-like manner. In order to attract additional immune cells to proximity of PTLD cells, based on the TÜ165 CAR, we moreover generated T cells redirected for universal cytokine-mediated killing (TRUCKs), which induce interleukin (IL)-12 release on target contact. METHODS: TÜ165-based CAR-T cells (CAR-Ts) and TRUCKs with inducible IL-12 expression in an all-in-one construct were generated. Functionality of the engineered cells was assessed in co-cultures with EBNA-3C-peptide-loaded, HLA-B*35-expressing K562 cells and EBV-infected B cells as PTLD model. IL-12, secreted by TRUCKs on target contact, was further tested for its chemoattractive and activating potential towards monocytes and natural killer (NK) cells. RESULTS: After co-cultivation with EBV target cells, TÜ165 CAR-Ts and TRUCKs showed an increased activation marker expression (CD137, CD25) and release of proinflammatory cytokines (interferon-γ and tumor necrosis factor-α). Moreover, TÜ165 CAR-Ts and TRUCKs released apoptosis-inducing mediators (granzyme B and perforin) and were capable to specifically lyse EBV-positive target cells. Live cell imaging revealed a specific attraction of TÜ165 CAR-Ts around EBNA-3C-peptide-loaded target cells. Of note, TÜ165 TRUCKs with inducible IL-12 showed highly improved effector functions and additionally led to recruitment of monocyte and NK cell lines. CONCLUSIONS: Our results demonstrate that TÜ165 CAR-Ts recognize EBV peptide/HLA complexes in a TCR-like manner and thereby allow for recognizing an intracellular EBV target. TÜ165 TRUCKs equipped with inducible IL-12 expression responded even more effectively and released IL-12 recruited additional immune cells which are generally missing in proximity of lymphoproliferation in immunocompromised PTLD patients. This suggests a new and promising strategy to specifically target EBV-infected cells while sparing and mobilizing healthy immune cells and thereby enable control of EBV-associated lymphoproliferation.

Effects of Lactate on One Class of Group III (CT3) Muscle Afferents.

A class of Group III muscle afferent neurons has branching sensory terminals in the connective tissue between layers of mouse abdominal muscles ("CT3 muscle afferents"). These sensory endings are both mechanosensitive and metabosensitive. In the present study, responses of CT3 afferents to lactate ions and changes in temperature were recorded. Raising muscle temperature from 32.7°C to 37°C had no consistent effects on CT3 afferent basal firing rate or responses to either von Frey hair stimulation or to an applied load. Superfusion with lactate ions (15 mM, pH 7.4) was associated with an increase in firing from 6 ± 0.7 Hz to 11.7 ± 6.7 Hz (14 units, n = 13, P < 0.05, P = 0.0484) but with considerable variability in the nature and latency of response. Reducing the concentration of extracellular divalent cations, which mimicked the chelating effects of lactate, did not increase firing. Raised concentrations of divalent cations (to compensate for chelation) did not block excitatory effects of lactate on CT3 afferents, suggesting that effects via ASIC3 were not involved. Messenger RNA for the G-protein coupled receptor, hydroxyl carboxylic acid receptor 1 (HCAR1) was detected in dorsal root ganglia and HCAR1-like immunoreactivity was present in spinal afferent nerve cell bodies retrogradely labeled from mouse abdominal muscles. HCAR1-like immunoreactivity was also present in axons in mouse abdominal muscles. This raises the possibility that some effects of lactate on group III muscle afferents may be mediated by HCAR1.

Thirty Mouse Strain Survey of Voluntary Physical Activity and Energy Expenditure: Influence of Strain, Sex and Day-Night Variation.

We measured indirect calorimetry and activity parameters, VO2 and VCO2 to extract respiratory exchange ratio (RER) and energy expenditure in both sexes of 30 inbred mouse strains of 6 genetic families at 9-13 weeks during one photophase and the subsequent scotophase. We observed a continuous distribution of all traits. While males had higher body weights than females, we observed no sex difference for food and water intake. All strains drank and fed more during the night even if they displayed no day-night difference in activity traits. Several strains showed absent or weak day-night variation in one or more activity traits and these included FVB and 129X1, males of 129S1, SWR, NZW, and SM, and females of SJL. In general females showed higher rearing and ambulatory activity with 6 and 9 strains, respectively, showing a sex difference. Fine motor movements, like grooming, showed less sex differences. RER underlied a strong day-night difference and no sex effect. Only FVB females and males of the RIIIS and SM strain had no day-night variation. Energy expenditure underlies a large day-night variation which was absent in SWR and in FVB females and RIIIS males. In general, female bodies had a tendency to higher energy expenditure values, which became a significant difference in C3H, MAMy, SM, DBA1, and BUB. Our data illustrate the diversity of these traits in male and female inbred mice and provide a resource in the selection of strains for future studies.

Design and Characterization of an "All-in-One" Lentiviral Vector System Combining Constitutive Anti-GD2 CAR Expression and Inducible Cytokines.

Genetically modified T cells expressing chimeric antigen receptors (CARs) so far have mostly failed in the treatment of solid tumors owing to a number of limitations, including an immunosuppressive tumor microenvironment and insufficient CAR T cell activation and persistence. Next-generation approaches using CAR T cells that secrete transgenic immunomodulatory cytokines upon CAR signaling, known as TRUCKs ("T cells redirected for universal cytokine-mediated killing"), are currently being explored. As TRUCKs were engineered by the transduction of T cells with two separate vectors, we developed a lentiviral modular "all-in-one" vector system that combines constitutive CAR expression and inducible nuclear factor of activated T cells (NFAT)-driven transgene expression for more efficient production of TRUCKs. Activation of the GD2-specific CAR via GD2+ target cells induced NFAT promoter-driven cytokine release in primary human T cells, and indicated a tight linkage of CAR-specific activation and transgene expression that was further improved by a modified NFATsyn promoter. As proof-of-concept, we showed that T cells containing the "all-in-one" vector system secrete the immunomodulatory cytokines interleukin (IL)12 or IL18 upon co-cultivation with primary human GD2+ tumor cells, resulting in enhanced effector cell properties and increased monocyte recruitment. This highlights the potential of our system to simplify application of TRUCK-modified T cells in solid tumor therapy.

Umbilical cord as a long-term source of activatable mesenchymal stromal cells for immunomodulation.

BACKGROUND: Mesenchymal stromal cells (MSCs) are used in over 800 clinical trials mainly due to their immune inhibitory activity. Umbilical cord (UC), the second leading source of clinically used MSCs, is usually cut in small tissue pieces. Subsequent cultivation leads to a continuous outgrowth of MSC explant monolayers (MSC-EMs) for months. Currently, the first MSC-EM culture takes approximately 2 weeks to grow out, which is then expanded and applied to patients. The initiating tissue pieces are then discarded. However, when UC pieces are transferred to new culture dishes, MSC-EMs continue to grow out. In case the functional integrity of these cells is maintained, later induced cultures could also be expanded and used for cell therapy. This would drastically increase the number of available cells for each patient. To test the functionality of MSC-EMs from early and late induction time points, we compared the first cultures to those initiated after 2 months by investigating their clonality and immunomodulatory capacity. METHODS: We analyzed the clonal composition of MSC-EM cultures by umbilical cord piece transduction using integrating lentiviral vectors harboring genetic barcodes assessed by high-throughput sequencing. We investigated the transcriptome of these cultures by microarrays. Finally, the secretome was analyzed by multiplexed ELISAs, in vitro assays, and in vivo in mice. RESULTS: DNA barcode analysis showed polyclonal MSC-EMs even after months of induction cycles. A transcriptome and secretome analyses of early and late MSC cultures showed only minor changes over time. However, upon activation with TNF-α and IFN-γ, cells from both induction time points produced a multitude of immunomodulatory cytokines. Interestingly, the later induced MSC-EMs produced higher amounts of cytokines. To test whether the different cytokine levels were in a therapeutically relevant range, we used conditioned medium (CM) in an in vitro MLR and an in vivo killing assay. CM from late induced MSC-EMs was at least as immune inhibitory as CM from early induced MSC-EMs. CONCLUSION: Human umbilical cord maintains a microenvironment for the long-term induction of polyclonal and immune inhibitory active MSCs for months. Thus, our results would offer the possibility to drastically increase the number of therapeutically applicable MSCs for a substantial amount of patients.