Cytotoxic Capacity of IL-15-Stimulated Cytokine-Induced Killer Cells Against Human Acute Myeloid Leukemia and Rhabdomyosarcoma in Humanized Preclinical Mouse Models

Allogeneic stem cell transplantation (allo-SCT) has become an important treatment modality for patients with high-risk acute myeloid leukemia (AML) and is also under investigation for soft tissue sarcomas. The therapeutic success is still limited by minimal residual disease (MRD) status ultimately leading to patients’ relapse. Adoptive donor lymphocyte infusions based on MRD status using IL-15-expanded cytokine-induced killer (CIK) cells may prevent relapse without causing graft-versus-host-disease (GvHD). To generate preclinical data we developed mouse models to study anti-leukemic- and anti-tumor-potential of CIK cells in vivo. Immunodeficient mice (NOD/SCID/IL-2Rγc−, NSG) were injected intravenously with human leukemic cell lines THP-1, SH-2 and with human rhabdomyosarcoma (RMS) cell lines RH41 and RH30 at minimal doses required for leukemia or tumor engraftment. Mice transplanted with THP-1 or RH41 cells were randomly assigned for analysis of CIK cell treatment. Organs of mice were analyzed by flow cytometry as well as quantitative polymerase chain reaction for engraftment of malignant cells and CIK cells. Potential of CIK cells to induce GvHD was determined by histological analysis. Tissues of the highest degree of THP-1 cell expansion included bone marrow followed by liver, lung, spleen, peripheral blood (PB), and brain. RH30 and RH41 engraftment mainly took place in liver and lung, but was also detectable in spleen and PB. In spite of delayed CIK cell expansion compared with malignant cells, CIK cells injected at equal amounts were sufficient for significant reduction of RH41 cells, whereas against fast-expanding THP-1 cells 250 times more CIK than THP-1 cells were needed to achieve comparable results. Our preclinical in vivo mouse models showed a reliable 100% engraftment of malignant cells which is essential for analysis of anti-cancer therapy. Furthermore our data demonstrated that IL-15-activated CIK cells have potent cytotoxic capacity against AML and RMS cells without causing GvHD

ErbB2 (HER2)-CAR-NK-92 cells for enhanced immunotherapy of metastatic fusion-driven alveolar rhabdomyosarcoma

IntroductionMetastatic rhabdomyosarcoma (RMS) is a challenging tumor entity that evades conventional treatments and endogenous antitumor immune responses, highlighting the need for novel therapeutic strategies. Applying chimeric antigen receptor (CAR) technology to natural killer (NK) cells may offer safe, effective, and affordable therapies that enhance cancer immune surveillance. MethodsHere, we assess the efficacy of clinically usable CAR-engineered NK cell line NK-92/5.28.z against ErbB2-positive RMS in vitro and in a metastatic xenograft mouse model.ResultsOur results show that NK-92/5.28.z cells effectively kill RMS cells in vitro and significantly prolong survival and inhibit tumor progression in mice. The persistence of NK-92/5.28.z cells at tumor sites demonstrates efficient antitumor response, which could help overcome current obstacles in the treatment of solid tumors.DiscussionThese findings encourage further development of NK-92/5.28.z cells as off-the-shelf immunotherapy for the treatment of metastatic RMS

Generation, identification and characterization of non-alloreactive T-lymphocytes for adoptive immunotherapy after allogeneic transplantation

Die hämatopoetische Stammzelltransplantation (HSCT) einer Megadosis immunoselektierter CD34+ allogener Stammzellen stellt seit Anfang der 90er Jahre eine allgemein anerkannte Therapieform maligner Erkrankungen oder angeborener Dysfunktionen des hämatopoetischen sowie des Immunsystems dar. Zu den ernstzunehmenden Komplikationen dieser Therapie zählen insbesondere die Transplantatabstoßung sowie eine verlängerte Periode der Immundefizienz mit der Gefahr lebensbedrohlicher Infektionen. In diesen Fällen kann die Applikation immunkompetenter T-Lymphozyten indiziert sein. Dieses Vorgehen ist jedoch mit einem erhöhten Risiko hinsichtlich der Entwicklung einer Graft versus Host Erkrankung (GVHD) verbunden. Aus diesem Grund entwickelten wir Strategien für eine spezifische Depletion alloreaktiver T-Zellen unter Bewahrung der adoptiven immunologischen Effekte, die durch die restlichen T-Lymphozyten hervorgerufen werden können. Um mit Hilfe einer Lymphozytenmischkultur (MLC) spezifisch aktivierte, alloreaktive T-Lymphozyten zu generieren, etablierten wir drei verschiedene Methoden der T-Zellstimulation unter Verwendung des Mediums X-Vivo15 mit einem Zusatz von 10% humanem AB-Serum. Hierzu wurden die T-Lymphozyten zusammen mit bestrahlten allogenen peripheren mononukleären Zellen (PMNC) (1), mit bestrahlten PMNC nach Übernachtinkubation mit Zytokinen (2) sowie mit bestrahlten Dendriten (3), monozytären Ursprungs kultiviert. Nach 72 Stunden exprimierten entsprechende T-Lymphozyten in unterschiedlichen Prozentsätzen die Aktivierungsantigen CD25, CD69, HLA-DR, CD71, CD95, CD161, CD28, CD90, CD8 und CD56. Dies zeigte, dass die Aktivierung alloreaktiver T-Lymphozyten in vitro möglich war und dass neben den verwendeten Antigenen weitere Aktivierungsmarker, wie z. B. das CD71 oder das CD161 Molekül für die immunomagnetische Depletion herangezogen werden könnten. Betrachtete man dabei die Aktivierungsmarker CD25, CD69 und HLA-DR, so wurden die höchsten Aktivierungsraten erreicht, wenn die Responder-T-Lymphozyten mit dendritischen Zellen ko-kultiviert worden waren, gefolgt von zytokinpräinkubierten PMNC und unbehandelten PMNC. Durch die immunomagnetischen Depletion gegen die Oberflächenmarker CD25, CD69 und HLA-DR mit Hilfe der Dynalbeads konnten die alloreaktiven T-Lymphozyten effizient depletiert werden. Dabei korrelierte die Effizienz der Depletion positiv mit der vorausgegangenen allogenen Aktivierung. Auch innerhalb der folgenden vierundzwanzigstündigen Restimulation konnte in jeder Versuchsreihe eine Reduktion der Alloreaktivität anhand einer verminderten Interferonsekretion, verglichen mit der Ausgangsituation vor Depletion bestätigt werden. Auch weitere fünf Tage später zeigte sich in dem Einbau des tritiummarkierten Thymidins in die zelleigene DNA, als Zeichen der proliferativen Zellaktivität, eine deutlich reduzierte Immunantwort nach Restimulation durch die ursprünglich verwendeten allogenen Stimulatoren. Die übrigen T-Zellen besaßen nachweislich weiterhin eine immunologische Aktivität in Gegenwart von „third party“ Zellen, Tetanus Toxoid, CMV-Antigen und PHA (Phythämagglutinin). Der Einsatz dieser Zellen im Rahmen einer adoptiven Immuntherapie nach allogener Stammzelltransplantation könnte neben einem wirksamen Schutz vor Virus- und Pilzinfektionen möglicherweise auch einen Schutz vor Abstoßungsreaktionen oder einem Rezidiv der Grunderkrankung bieten.Hematopoietic stem cell transplantation is successfully used to treat leukemia and many other hematological and immunological disorders. After allogeneic transplantation of highly purified CD34+ hematopoietic progenitor cells, delayed immune reconstitution may result in increased rates of leukemia relapse, graft rejection and severe infections. To overcome this problems donor lymphocyte infusions (DLI) could be given. However, T-cell infusions are associated with the risk of a graft-versus-host reaction, which may lead to a life-threatening disease (GVHD). We therefore investigated a depletion strategy based on the selective immunomagnetic removal of alloreactive T-cells while retaining non-alloreactive T-cells capable of graft-versus-leukemia (GVL) and antiviral activity. In order to generate specifically activated alloreactive T-cells in a mixed-lymphocyte-culture (MLC) we established three different methods of T-cell-stimulation. Therefore responder cells were cocultured with irradiated mononuclear cells, with irradiated peripheral blood cells pretreated with TNF-alpha and IFN-gamma and with irradiated monocyte/macrophage-derived-dentritic-cells. Activation kinetics of alloreactive T-cells were analyzed by FACS based on the expression of the activation markers CD25, CD69 and HLA-DR. Analyzing the coexpression of these activation markers upregulation started as early as 24h after allogeneic stimulation and peaked at 72h. In addition to the previous mentioned antigens other markers like i.e. CD71 and CD161 also seemed to be suitable for phenotypic analyses and subsequent immunomagnetic removal of alloreactive T-cells. After a defined period of 72h depending on the activation kinetics alloreactive responder cells were immunomagnetically depleted using antibodies against the activation antigens CD25, CD69 and HLA-DR and magnetic particles (Dynal). Alloreactive cells were minimized to less than 1% of CD25, CD69 and HLA-DR positive cells, which was determined by FACS-analyses using a secondary GAM-antibody. The efficiency of depletion correlated with the density of the activation antigens on alloreactive T-cells and therefore depended on the previous method of allogeneic activation. Within the next 24h of allogeneic restimulation alloreactivity still remained reduced. This was ascertained by a diminished IFN-gamma-release measured by IFN-gamma-elispot-assays and secretion-assays. Even 120h after depletion proliferation of alloreactive cells was strongly reduced, which was shown by H3-thymidine incorporation, whereas the immunological response against i.e. third party, tetanus toxoid and CMV-antigen was almost preserved. Depletion of alloreactive cells represents a promising way to avoid graft-versus-host disease throughout adoptive immunotherapy

Aminoglycoside block of P2X2 receptors heterologously expressed in Xenopus laevis oocytes

Aminoglycosides are polycationic antibiotics that have been shown to block a variety of cation channels. The inhibitory effect of externally applied aminoglycosides on P2X2 receptor currents was examined after heterologous expression in Xenopus laevis oocytes using the two-electrode voltage-clamp technique. All of the aminoglycosides tested inhibited the ATP-evoked responses with potencies ranging from 71 μM to 2 mM (IC50 values). The ranked order of potency was streptomycin > gentamicin > neomycin > paromomycin > kanamycin. The inhibition of P2X receptor currents was independent of the ATP concentration used for the activation, which is compatible with a noncompetitive mechanism. The inhibition was voltage-dependent and was reduced at more positive membrane potentials. To examine whether the current block was dependent on the receptor conformation, the aminoglycoside effect on a non-desensitizing P2X2-X1 receptor chimera was analyzed. The results from these measurements suggest that inhibition is caused by an open pore block that locks the P2X receptor chimera in an open nonconducting state from which the agonist dissociation is slow. We also demonstrate that the P2X2-X1 chimera can serve as a tool to directly test whether an antagonist acts competitively or not

In-vitro influence of mycophenolate mofetil (MMF) and Ciclosporin A (CsA) on cytokine induced killer (CIK) cell immunotherapy

Background: Cytokine-induced-killer (CIK) cells are a promising immunotherapeutic approach for impending relapse following hematopoietic stem cell transplantation (HSCT). However, there is a high risk for treatment failure associated with severe graft versus host disease (GvHD) necessitating pharmaceutical intervention post-transplant. Whether immunosuppression with mycophenolate mofetil (MMF) or Ciclosporin A (CsA) influences the cytotoxic effect of CIK cell immunotherapy is still an open issue. Methods: CIK cells were generated from PBMC as previously described followed by co-incubation with mycophenolic acid (MPA) or CsA. Proliferation, cytotoxicity and receptor expression were investigated following short- (24 h), intermediate- (3 days) and long-term (7 days) MPA incubation with the intention to simulate the in vivo situation when CIK cells were given to a patient with relevant MPA/CsA plasma levels. Results: Short-term MPA treatment led to unchanged proliferation capacity and barely had any effect on viability and cytotoxic capability in vitro. The composition of CIK cells with respect to T-, NK-like T- and NK cells remained stable. Intermediate MPA treatment lacked effects on NKG2D, FasL and TRAIL receptor expression, while an influence on proliferation and viability was detectable. Furthermore, long-term treatment significantly impaired proliferation, restricted viability and drastically reduced migration-relevant receptors accompanied by an alteration in the CD4/CD8 ratio. CD3+CD56+ cells upregulated receptors relevant for CIK cell killing and migration, whereas T cells showed the most interference through significant reductions in receptor expression. Interestingly, CsA treatment had no significant influence on CIK cell viability and the cytotoxic potential against K562. Conclusions: Our data indicate that if immunosuppressant therapy is indispensable, efficacy of CIK cells is maintained at least short-term, although more frequent dosing might be necessary