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Multiple myeloma (MM) is a plasma cell disease with a preferential bone marrow (BM) tropism. Enforced expression of tissue-specific chemokine receptors has been shown to successfully guide adoptively-transferred CAR NK cells towards the malignant milieu in solid cancers, but also to BM-resident AML and MM. For redirection towards BM-associated chemokine CXCL12, we armored BCMA CAR-NK-92 as well as primary NK cells with ectopic expression of either wildtype CXCR4 or a gain-of-function mutant CXCR4R334X. Our data showed that BCMA CAR-NK-92 and -primary NK cells equipped with CXCR4 gained an improved ability to migrate towards CXCL12 in vitro. Beyond its classical role coordinating chemotaxis, CXCR4 has been shown to participate in T cell co-stimulation, which prompted us to examine the functionality of CXCR4-cotransduced BCMA-CAR NK cells. Ectopic CXCR4 expression enhanced the cytotoxic capacity of BCMA CAR-NK cells, as evidenced by the ability to eliminate BCMA-expressing target cell lines and primary MM cells in vitro and through accelerated cytolytic granule release. We show that CXCR4 co-modification prolonged BCMA CAR surface deposition, augmented ZAP-70 recruitment following CAR-engagement, and accelerated distal signal transduction kinetics. BCMA CAR sensitivity towards antigen was enhanced by virtue of an enhanced ZAP-70 recruitment to the immunological synapse, revealing an increased propensity of CARs to become triggered upon CXCR4 overexpression. Unexpectedly, co-stimulation via CXCR4 occurred in the absence of CXCL12 ligand-stimulation. Collectively, our findings imply that co-modification of CAR-NK cells with tissue-relevant chemokine receptors affect adoptive NK cell therapy beyond improved trafficking and retention within tumor sites.

Abstract Background Lameness in cattle is a major health problem and causes great economic losses. Carpal injury is a common cause of forelimb lameness in cattle. Radiography and/or ultrasonography of the carpus is a challenge due to complex anatomy of the joint. Additional imaging using computed tomography (CT) or magnetic resonance imaging (MRI) may be indispensable for reaching a decisive diagnosis. Precise evaluation of the clinical CT and MRI images necessitates an in-depth knowledge of the normal CT and MRI tissue variants. Therefore, our purpose was to provide a detailed description of the normal CT and MRI appearance of the osseous and soft tissue structures of twelve cadaveric bovine carpi using CT and 3 Tesla MRI. Carpi were frozen, transected in sagittal, dorsal and transverse planes then adjoined to their corresponding CT and MRI images. Results The clinically significant articular and peri-articular structures of the bovine carpus were identified and characterized on the CT and MRI images. CT images provided a remarkable delineation of the cortical, subchondral, and cancellous bone. The high-field 3 Tesla MRI offered high definition and distinction of the delicate soft tissues of the bovine carpus. Conclusions 3 Tesla high-field MRI offers new opportunities in soft tissue tomography but cannot be compared with CT in terms of bone imaging. Clinicians have to determine whether CT, MRI or both imaging techniques are required in clinical situations.

The advent of CAR T cells targeting CD19 or BCMA on B cell neoplasm demonstrated remarkable efficacy, but rapid relapses and primary refractoriness remains challenging. A leading cause of CAR T cell failure is their lack of expansion and limited persistence. Long-lived, self-renewing multipotent T memory stem cells (TSCM) and T central memory cells (TCM) likely sustain superior tumor regression, but their low frequencies in blood from cancer patients impose a major hurdle for clinical CAR T production. We designed a clinically compliant protocol for generating BCMA CAR T cells starting with increased TSCM/TCM cell input. A CliniMACS Prodigy process was combined with flow cytometry-based enrichment of CD62L+CD95+ T cells. Although starting with only 15% of standard T cell input, the selected TSCM/TCM material was efficiently activated and transduced with a BCMA CAR-encoding retrovirus. Cultivation in the presence of IL-7/IL-15 enabled the harvest of CAR T cells containing an increased CD4+ TSCM fraction and 70% TSCM cells amongst CD8+. Strong cell proliferation yielded cell numbers sufficient for clinical application, while effector functions were maintained. Together, adaptation of a standard CliniMACS Prodigy protocol to low input numbers resulted in efficient retroviral transduction with a high CAR T cell yield.

Abstract Background Long-term cyclic tensile testing with equine palmar/plantar tendons have not yet been performed due to problems in fixing equine tendons securely and loading them cyclically. It is well established that the biomechanical response of tendons varies during cyclic loading over time. The aim of this study was to develop a clamping device that enables repetitive cyclic tensile testing of equine superficial digital flexor tendon for at least 60 loading cycles and for 5 min. Results A novel cryo-clamp was developed and built. Healthy and collagenase-treated pony SDFTs were mounted in the custom-made cryo-clamp for the proximal tendon end and a special clamping device for the short pastern bone (os coronale). Simultaneously with tensile testing, we used a biplanar high-speed fluoroscopy system (FluoKin) to track tendon movement. The FluoKin system was additionally validated in precision measurements. During the cyclic tensile tests of the SDFTs, the average maximal force measured was 325 N and 953 N for a length variation of 2 and 4 % respectively. The resulting stress averaged 16 MPa and 48 MPa respectively, while the modulus of elasticity was 828 MPa and 1212 MPa respectively. Length variation of the metacarpal region was, on average, 4.87 % higher after incubation with collagenase. The precision of the FluoKin tracking was 0.0377 mm, defined as the standard deviation of pairwise intermarker distances embedded in rigid bodies. The systems accuracy was 0.0287 mm, which is the difference between the machined and mean measured distance. Conclusion In this study, a good performing clamping technique for equine tendons under repetitive cyclic loading conditions is described. The presented cryo-clamps were tested up to 50 min duration and up to the machine maximal capacity of 10 kN. With the possibility of repetitive loading a stabilization of the time-force-curve and changes of hysteresis and creep became obvious after a dozen cycles, which underlines the necessity of repetitive cyclical testing. Furthermore, biplanar high-speed fluoroscopy seems an appropriate and highly precise measurement tool for analysis of tendon behaviour under repetitive load in equine SDFTs.

Insight into processes that determine CD8+ T cell memory formation has been obtained from infection models. These models are biased toward an inflammatory milieu and often use high-avidity CD8+ T cells in adoptive-transfer procedures. It is unclear whether these conditions mimic the differentiation processes of an endogenous repertoire that proceed upon noninflammatory conditions prevailing in premalignant tumor lesions. We examined the role of cytolytic capacity on CD8+ T cell fate decisions when primed by tumor cells or by minor histocompatibility antigen–mismatched leukocytes. CD8+ memory commitment was analyzed in Ebag9-deficient mice that exhibited enhanced tumor cell lysis. This property endowed Ebag9–/– mice with extended control of Tcl-1 oncogene–induced chronic lymphocytic leukemia progression. In Ebag9–/– mice, an expanded memory population was obtained for anti-HY and anti–SV-40 T antigen–specific T cells, despite unchanged effector frequencies in the primary response. By comparing the single-cell transcriptomes of CD8+ T cells responding to tumor cell vaccination, we found differential distribution of subpopulations between Ebag9+/+ and Ebag9–/– T cells. In Ebag9–/– cells, these larger clusters contained genes encoding transcription factors regulating memory cell differentiation and anti-apoptotic gene functions. Our findings link EBAG9-controlled cytolytic activity and the commitment to the CD8+ memory lineage.

Abstract Background Despite clinical importance and frequent occurrence of sinus disease, little is known about the size of paranasal sinuses and their communication in ponies and small horses. To examine the shape and volume of the paranasal sinuses and evaluate the sinonasal communication, three-dimensional (3D) reconstructions of computed tomography (CT) datasets of 12 healthy adult Shetland ponies were performed and analysed. Linear measurements of head length and width were taken. Using semi-automatic segmentation, 3D-models of all sinus compartments were created. Volumetric measurement of the seven sinus compartments were conducted and statistical analysis was performed. Sinus volumes were compared between the left and right sinuses and the relation to age and head size was evaluated. Results Structure and shape of the paranasal sinus system in Shetland ponies was similar to that of large horses. All seven sinus compartments on each side of the head were identified (rostral maxillary sinus, ventral conchal sinus, caudal maxillary sinus, dorsal conchal sinus, middle conchal sinus, frontal sinus, sphenopalatine sinus). The existence of a bilateral cranial and a caudal system formed by a maxillary septum was visible in all 12 individuals. The volumetric sizes of the left and right sinuses did not differ significantly (p > 0.05). A positive correlation between the size of the paranasal sinuses and the head length was shown. A relation between sinus volumes and age could not be proved in adult ponies aged > six years. Communication between single sinus compartments was identified. Furthermore, communication with the nasal cavity over the nasomaxillary aperture (Apertura nasomaxillaris) and a common sinonasal channel (Canalis sinunasalis communis) as well as its splitting up into a rostral and a caudolateral channel could be seen. Examination of the sinonasal communication was challenging and only a descriptive evaluation was possible. Conclusions Our findings concerning the size, shape and volumetric dimensions of Shetland pony CT images could help improve CT interpretation of abnormal clinical cases as well as aiding clinicians to develop and select appropriate instruments for medical inspection and treatments.

The superficial digital flexor tendon (SDFT) is the most frequently injured structure of the musculoskeletal system in sport horses and a common cause for early retirement. This project’s aim was to visualize and measure the strain of the sound, injured, and healing SDFTs in a pony during walk and trot. For this purpose, biplanar high-speed fluoroscopic kinematography (FluoKin), as a high precision X-ray movement analysis tool, was used for the first time in vivo with equine tendons. The strain in the metacarpal region of the sound SDFT was 2.86% during walk and 6.78% during trot. When injured, the strain increased to 3.38% during walk and decreased to 5.96% during trot. The baseline strain in the mid-metacarpal region was 3.13% during walk and 6.06% during trot and, when injured, decreased to 2.98% and increased to 7.61%, respectively. Following tendon injury, the mid-metacarpal region contributed less to the overall strain during walk but showed increased contribution during trot. Using this marker-based FluoKin technique, direct, high-precision, and long-term strain measurements in the same individual are possible. We conclude that FluoKin is a powerful tool for gaining deeper insight into equine tendon biomechanics.

Treatment of tendon disease with multipotent mesenchymal stromal cells (MSC) is a promising option to improve tissue regeneration. To elucidate the mechanisms by which MSC support regeneration, longitudinal tracking of MSC labelled with superparamagnetic iron oxide (SPIO) by magnetic resonance imaging (MRI) could provide important insight. Nine equine patients suffering from tendon disease were treated with SPIO-labelled or nonlabelled allogeneic umbilical cord-derived MSC by local injection. Labelling of MSC was confirmed by microscopy and MRI. All animals were subjected to clinical, ultrasonographical, and low-field MRI examinations before and directly after MSC application as well as 2, 4, and 8 weeks after MSC application. Hypointense artefacts with characteristically low signal intensity were identified at the site of injection of SPIO-MSC in T1- and T2∗-weighted gradient echo MRI sequences. They were visible in all 7 cases treated with SPIO-MSC directly after injection, but not in the control cases treated with nonlabelled MSC. Furthermore, hypointense artefacts remained traceable within the damaged tendon tissue during the whole follow-up period in 5 out of 7 cases. Tendon healing could be monitored at the same time. Clinical and ultrasonographical findings as well as T2-weighted MRI series indicated a gradual improvement of tendon function and structure.