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Glioblastoma (GBM) is the most frequent and lethal malignant brain tumor in adults. It emerges with an incidence of 3.2 per 100.000 in the US and 3.91 in Europe. Today, standard treatment after diagnosis consists of surgical removal of tumor tissue, followed by radiation therapy and adjuvant chemotherapy using temozolomide. Even after this rigorous therapy, patients show a median overall survival of only 15.6 months or 20.5 months when the tumor is additionally treated with so-called tumor treating fields. GBM is characterized by molecular heterogeneity within the same patient but also between different patients, which impedes development of novel therapeutics. During the last decades various immunotherapies including (multi-epitope) peptide vaccines, oncolytic viruses or immune checkpoint inhibitors against GBM were tested in small clinical studies, but failed to show a benefit in large studies. A novel kind of immunotherapies that showed great success in hematological tumors so far, is based on chimeric antigen receptors (CAR). These synthetic receptors can be introduced into immune cells to retarget their function towards tumor cells, independently of the major histocompatibility complex (MHC) that is often down regulated by tumors for immune evasion. A large hurdle for treatment of GBM using immunotherapies such as CAR-T cells, is antigen heterogeneity that limits the effect of therapies against single targets and renders the need for discovery of novel targets to enable treatment of a wide variety of patients with high success. Analyzing publicly available data and performing RT-qPCR experiments with RNA isolated from GBM tissue of a local cohort of patients, overexpression of two candidate GBM antigens, namely TMEM158 and PTPRZ1 were observed. Overexpression of both antigens in GBM in comparison to normal brain tissue and low-grade gliomas (only TMEM158) was revealed. In addition, a negative correlation between expression and patient survival was detected, as well as a correlation between TMEM158 and CD44 expression, the latter being a marker for GBM stem cells and the mesenchymal GBM subtype. Induction of chemoresistance by TMEM158 seems likely for GBM, since this was already discovered for several other tumor entities. Protein expression of TMEM158 was confirmed by Western blot analysis of different GBM cell lines. Since cell surface expression of a target protein is a prerequisite for targeting by a CAR-therapy, the expression of TMEM158 on cells from GBM cell lines was analyzed by flow cytometry. For this analysis a fluorescence-labeled peptide, based on sequence information of a known naturally occurring TMEM158 ligand (BINP) was designed. Binding to T98G and U-87 MG was observed, while only very low binding to the neuroblastoma cell line SH-SY5Y was seen in flow cytometry. Partial knockdown of TMEM158 was achieved using DsiRNAs, followed by Western blot (antibody staining) and flow cytometry (peptide staining), confirming the specificity of binding detectable by both methods. A recombinant fusion protein, consisting of the extracellular part of TMEM158 and a human Fc-antibody fragment was produced in 293T cells by transient transfection of an expression vector. The expected size of the protein produced was confirmed by Western blot. Furthermore, binding of the BINP-peptide to the recombinant protein was analyzed and compared to a scrambled BINP-peptide. In these experiments specific binding of the BINP-peptide was observed, also indicating the functionality of the recombinant protein. Next, CAR-constructs were designed using the original sequence information from BINP as binding domain and additional variants with amino acid exchanges at different positions. Significant cytotoxicity of all BINP-CAR-T cells was observed against T98G, which showed highest binding of BINP when analyzed by flow cytometry. A BINP-CAR version in which phenylalanine 11 was exchanged with alanine (BINP-F11A-CAR) showed significantly higher cytotoxicity against T98G than the BINP-CAR containing the original BINP sequence (BINP-WT-CAR). Against the U-87 MG cell line, only a version of the BINP-CAR containing an RGD- (arginine-glycine-aspartic acid) motif showed significant cytotoxicity. RGD-motifs are known to bind integrins like αVβ3, which was abundantly present on this cell line, as it was confirmed by flow cytometry within this work. Using this BINP-RGD-CAR version, targeting of both antigens at the same time seems possible. No significant cytotoxicity of the different CAR versions was observed against the TMEM158- and αVβ3-low cell line SH-SY5Y. In conclusion, overexpression of TMEM158 and PTPRZ1 and their negative influence on survival of patients, as found in recent literature, was confirmed for glioblastoma. Significantly higher expression of TMEM158 in GBM in comparison to low-grade gliomas as well as the correlation with CD44 hint at an association of TMEM158 with the aggressive phenotype of GBM. For all of these reasons, targeting of TMEM158 appears to be very feasible. Cytotoxicity of the produced BINP-CAR-T cells, which are the first CAR-T cells targeting TMEM158 so far, was demonstrated against GBM cells. Additional to cytotoxicity of the CAR-T cells, other in vitro assays and in vivo models should be utilized to determine more aspects of CAR-T cell function, in the future. For example, proliferation, cytokine release, invasion of tumor tissue, and inactivation of CAR-T cells by the tumor milieu should be quantified. To estimate how many patients could benefit from a therapy against it, percentage of patients and distribution within the tumors should be determined.

Yes / Alopecia areata (AA) is an autoimmune hair loss disease with infiltration of proinflammatory cells into hair follicles. Current therapeutic regimens are unsatisfactory mainly because of the potential for side effects and/or limited efficacy. Here we report that cultured, transduced fibroblasts, which express the immunomodulatory molecule indoleamine 2,3-dioxygenase (IDO), can be applied to prevent hair loss in an experimental AA model. A single intraperitoneal (IP) injection of IDO-expressing primary dermal fibroblasts was given to C3H/HeJ mice at the time of AA induction. While 60–70% of mice that received either control fibroblasts or vehicle injections developed extensive AA, none of the IDO-expressing fibroblast-treated mice showed new hair loss up to 20 weeks post injection. IDO cell therapy significantly reduced infiltration of CD4+ and CD8+ T cells into hair follicles and resulted in decreased expression of TNF-α, IFN-γ and IL-17 in the skin. Skin draining lymph nodes of IDO fibroblast-treated mice were significantly smaller, with more CD4+ CD25+ FoxP3+ regulatory T cells and fewer Th17 cells than those of control fibroblast and vehicle-injected mice. These findings indicate that IP injected IDO-expressing dermal fibroblasts can control inflammation and thereby prevent AA hair loss. / Canadian Institutes of Health Researches (Funding Reference Number: 134214 and 136945).

Yes / Introduction: Skin ageing involves senescent fibroblast accumulation, disturbance in extracellular matrix (ECM) homeostasis, and decreased collagen synthesis. Objective: to assess a cell therapy product for aged skin (RCS-01; verum) consisting of ~25 × 106 cultured, autologous cells derived from anagen hair follicle non-bulbar dermal sheath (NBDS). Methods: For each subject in the verum group, 4 areas of buttock skin were injected intradermally 1 or 3 times at monthly intervals with RCS-01, cryomedium, or needle penetration without injection; in the placebo group RCS-01 was replaced by cryomedium. The primary endpoint was assessment of local adverse event profiles. As secondary endpoints, expression of genes related to ECM homeostasis was assessed in biopsies from randomly selected volunteers in the RCS-01 group taken 4 weeks after the last injection. ­Results: Injections were well tolerated with no severe adverse events reported 1 year after the first injection. When compared with placebo-treated skin, a single treatment with RCS-01 resulted in a significant upregulation of TGFβ1, CTGF, COL1A1, COL1A2, COL3A1, and lumican mRNA expression. Limitations: The cohort size was insufficient for dose ­ranging evaluation and subgroup analyses of efficacy. Conclusions: RCS-01 therapy is well tolerated and associated with a gene expression response consistent with an improvement of ECM homeostasis. / Replicel Life Sciences Inc, Vancouver, Canada.

Chronisches Nierenversagen kann zu einem endgültigen Funktionsverlust der Nieren führen. Nierenersatztherapien bleiben dabei die einzigen lebensrettenden Behandlungsmaßnahmen. Der globale Bedarf an Nierenorganen übersteigt jedoch bei weitem die Anzahl verfügbarer Spenderorgane. Humane induzierte pluripotente Stammzellen (hiPSC) stellen eine vielversprechende Quelle für die Generierung funktioneller Nierenzellen dar. Die hiPSC-abgeleiteten Nierenzellen könnten zukünftig für zelluläre Ersatztherapien verwendet werden. Innerhalb dieser Arbeit sollten die aus hiPSC-abgeleiteten Nierenzellen auf ihre Immunogenität überprüft werden. Dafür wurden hiPSC-Linien, reprogrammiert aus primären Urinzellen gesunder Spender, generiert. Die hiPSC wurden daraufhin in renale Vorläuferzellen (IM-Zellen) und proximale Tubuluszellen (PT-Zellen) differenziert. Die immunphänotypische Analyse ergab eine generell verminderte Expression immun-relevanter Gene verglichen zu adulten Urinzellen. Für die Immunigenitätsbestimmung wurden die Nierenzellen jeweils mit autologen oder allogenen Zellen des peripheren Blutes (PBMCs) kokultiviert. Die IM- sowie PT-Zell-Kokulturen mit PBMC gesunder Spender sowie von Patienten mit diabetischer Nephropathie ergaben keine Proliferation autoreaktiver sowie allogener T-Zellen, im Vergleich zu primäre Urinzellen mit gleichen HLA-Merkmalen. Des Weiteren wiesen IM- und PT-Zellen aktive immunsupprimierende Eigenschaften auf. Diese konnten in langzeit-kultivierten PT-Zellen nicht mehr beobachtet werden. Zusätzlich zeigten die hiPSC-abgeleiteten Nierenzellen eine Anfälligkeit gegenüber autologe sowie allogene natürliche Killerzellen (NK-Zellen). Diese Studie ergab erstmalig Einblicke in die Dynamiken der T- und NK-Zell-Antworten renal differenzierter hiPSC. Die hier gewonnenen Erkenntnisse können für die Entwicklung zukünftiger Therapien beitragen, um eine sichere Transplantation von hiPSC-abgeleiteten Nierenzellen, selbst im allogenen System, zu ermöglichen. / Kidneys are essential for numerous vital processes. Chronic damage can lead to end stage renal disease with the requirement of kidney replacement therapies. Kidneys are nowadays the most frequently transplanted human organs, however, the transplant demand is far exceeding the number of available donations. Human induced pluripotent stem cells (hiPSC) represent a promising alternative approach for the generation of functional renal differentiated cells suitable for autologous cell replacement therapies (CRT). Yet, the immunogenic potential of hiPSC and their progenies are among the major concerns. This study aimed to analyze immunogenic effects of hiPSC-derived renal cells in autologous and allogeneic settings in vitro. Therefor, primary urinary cells (pUC) from healthy donors were reprogrammed into hiPSC, which were differentiated into renal progenitors (IMC) and into proximal tubular cells (PTC). Immune-phenotypic characterization revealed overall reduced expression of immune-relevant genes in hiPSC and renal derivatives compared to pUC. Co-culture experiments of IMC or PTC with either autologous or allogeneic peripheral blood mononuclear cells of healthy donors or patients with diabetic nephropathy revealed no induction of T cell proliferation in comparison to pUC cells with the same HLA-types. IMC and PTC showed immunomodulatory effects on allogeneic T cell proliferation. These immunosuppressive capacities were lost in long-term cultivated PTC. hiPSC-derived renal cells showed to elicit autologous as well as for allogeneic natural killer cells (NK cells). This study provided insights about the immunogenic properties of hiPSC-derived renal cells. In vitro experiments revealed new hints about the dynamics of T- and NK cell responses dependent on the differentiation status of hiPSC-derived renal cells. These data show translational potential for the development of future CRT strategies aiming safe transplantation of hiPSC-derived renal cells across HLA barriers.

Le diabète, caractérisé notamment par une atteinte neuro-vasculaire, représente aujourd’hui un réel problème de santé publique. Cependant plus de 30% des patients, ne peuvent bénéficier des traitements actuels (chirurgie et pharmacothérapie) d’où, la nécessité de développer de nouvelles approches thérapeutiques innovantes. L’objectif de ce travail a été de caractériser un modèle murin de neuro-angiopathie diabétique, représentatif de la pathologie humaine puis, d’évaluer les effets des molécules guidance neuronale dans la récupération de la fonction neuro-vasculaire. Dans un premier temps, nous avons mis en place un modèle et des techniques exploratoires de l’étude de la fonction neuro-vasculaire chez les souris diabétiques. Nous avons observé que suite à l’ischémie du membre inférieur, la revascularisation est altérée chez les animaux diabétiques par comparaison aux animaux non-diabétiques. A ce défaut de la fonction vasculaire, s’ajoute également un défaut de la fonction nerveuse. En effet, les paramètres électrophysiologiques, le flux sanguin nerveux, le nombre de capillaires dans le nerf sciatique et la régénération nerveuse sont fortement impactés chez les animaux diabétiques. Afin de palier au défaut de revascularisation et/ou de régénération nerveuse, nous avons proposé que le ciblage thérapeutique des molécules de guidance neuronale, éphrine-B2 et Sémaphorine-3A (Séma-3A) améliorerait la fonction neuro-vasculaire. Après ischémie, les cellules mononuclées du sang périphérique (CM-SP) provenant de patients diabétiques ou de sujets contrôles, traitées préalablement avec l’éphrine-B2/Fc, ont été ensuite injectées aux animaux diabétiques. Une nette amélioration de la revascularisation a été observée dans les groupes d’animaux traités avec l’éphrine-B2/Fc par rapport aux animaux non-traités ou traités avec des CM-SP non stimulées. Enfin, nous avons administré un inhibiteur de Séma-3A, SM-345431 (Vinaxanthone) à des animaux contrôles et diabétiques après ischémie du membre inférieur. Ce qui nous a permis de montrer que cette inhibition améliore significativement la revascularisation et régule notamment la voie de signalisation des MAPK, Erk1/2 et p38 ainsi que, les niveaux de VEGF et de TGF-β1. Dans l’ensemble, l’effet sur la fonction nerveuse périphérique n’a pas montré de différences majeures entre les groupes traités et non-traités. En somme, nos travaux ont permis de montrer que la thérapie cellulaire utilisant les CM-SP stimulées avec l’éphrine-B2/Fc, ou bien l’injection intramusculaire d’un inhibiteur de Séma-3A, à visée pro-angiogénique et/ou pro-neurorégénérative, apparaît comme une stratégie prometteuse et susceptible d’améliorer la qualité de vie des malades atteints de diabète. / Diabetes, characterized by neuro-vascular damages, is a real public health problem. More than 30% of diabetic patients can benefit from existing therapies (surgery and drug therapy), therefore, the need to develop new innovative therapeutic approaches. The objective of this work was to characterize a diabetic neuro-angiopathy mouse model, reproducible of human pathology and to assess the effects of neuronal guidance molecules in recovery of the neuro-vascular function. Firstly, we characterized a model and exploratory techniques to study the neuro-vascular functions in diabetic mice. After hindlimb ischemia induction, we observed that revascularization were impaired in diabetic animals compared to non diabetic animals. This alteration is associated of the nerve function defect. Indeed, the electrophysiological parameters, nerve blood flow, the number of capillaries in sciatic nerve and nerve regeneration are strongly affected in diabetic animals. Then, to rescue the revascularization or nerve regeneration, we proposed that the therapeutic targeting of molecules of neuronal guidance, ephrin-B2 and Semaphorin-3A (Sema-3A) would improve neuro-vascular functions. After ischemia induction, peripheral mononuclear blood mononuclear cells (PBMNC) from diabetic patients or subjects controls, pre-treated with ephrin-B2/Fc, were then injected in diabetic animals. A significant improvement of revascularization was observed in both groups, animals treated with ephrin-B2/Fc compared to untreated animals or treaties with PBMNC unstimulated. Finally, after hindlimb ischemia, we administrated a pharmacological inhibitor of Sema-3A, the SM-345431 (Vinaxanthone) in diabetic and control animals. We showed this selective inhibition significantly improved the post-ischemic revascularization, regulates the MAPK Erk1/2 and p38 signaling pathway, increased the levels of CXCL12, VEGF and TGF-β1. In our experiment conditions, we did not observe the significant effects of SM-345431 on peripheral nerve function between treated and untreated animal groups. Finally, our work showed that cell therapy using PBMNC stimulated with ephrin-B2/Fc, or the intramuscular injection of Sema-3A inhibitor referred to pro-angiogenic and/or pro-neuroregenerative, appears as a strategy promising and likely to improve the quality of life of diabetic patients.