Einfluss von Natürlichen Killer (NK) Zellen auf den Graft versus Leukämie-, Tumor-Effekt und die Immunrekonstitution bei pädiatrischen Patienten mit malignen Erkrankungen nach haploidenter Stammzelltransplantation

Die adoptive Immuntherapie mit hochaufgereinigten NK-Zellen bei pädiatrischen Patienten mit malignen Erkrankungen nach haploidenter SZT ist eine mögliche Therapieoption, um einen verstärkten GvL/GvT-Effekt zu bewirken und möglicherweise die Immunregeneration zu fördern. Als schwerwiegende Nebenwirkung ist bisher noch nicht eindeutig belegt, ob neben T-Zellen auch NK-Zellen in der Lage sind, eine GvHD auszulösen. In der Frankfurter Universitätskinderklinik wurden 7 Patienten (4xALL, 1xAML, 1xRMS IV und 1xM. Hodgkin) mit hochaufgereinigten unstimulierten NK-Zellen und 3 Patienten (3xNB IV) mit IL-2 stimulierten NK-Zellen nach haploidenter SZT behandelt. Im Rahmen dieser Arbeit wurde in vitro untersucht, ob NK-Zellen durch die Stimulierung mit IL-2 einen gesteigerten GvL/T-Effekt aufweisen. Es wurden NK-Zellen von 5 verschiedenen gesunden Spendern (2 im Rahmen von Validierungsläufen und 3 zur Behandlung der 3 Patienten mit NB) zunächst immunomagnetisch im klinischen Maßstab mittels CD3-Depletion und darauffolgender CD56-Selektion aufgereinigt. Danach erfolgte die Aktivierung mit IL-2 (Proleukin®S) über 14 Tage unter GMP. Während sich die NK-Zellen aller Spender nach Aufreinigung in eine kleine Population CD56+CD16- immunregulatorischer NK-Zellen (2,3 bis 7,1 %) und eine große Population zytotoxischer CD56+CD16+ NK-Zellen (92,9 bis 97,7 %) unterteilen ließen, zeigte sich nach IL-2 Stimulierung ein heterogenes Bild von CD16+ zu CD16- NK-Zellen. Durch die 9-tägige IL-2 Stimulierung vergrößerte sich der Anteil KIRnegativer NK-Zellen. Es konnte auf den NK-Zellen aller Spender gezeigt werden, dass durch die IL-2 Stimulierung wichtige Rezeptoren (NKG2D, NCR), die für ein hohes zytotoxisches Potential stehen, verstärkt auf der NK-Zelloberfläche exprimiert wurden. Die gesteigerte Zytokinproduktion der IL-2 stimulierten NK-Zellen untermauerte die Funktionalität der ex vivo stimulierten NK-Zellen und dies konnte in funktionalen Assays, die die zytotoxische Aktivität von NK-Zellen belegen, bewiesen werden. Durch die IL-2 Stimulierung der NK-Zellen konnte die Killing Aktivität gleichmäßig auf über 90 % gesteigert werden. Interessanterweise wies Spender A bei den funktionellen und phänotypischen Analysen eine Sonderrolle auf. Die NK-Zellen dieses Spenders zeigten bereits vor IL-2 Stimulierung eine hohe Zytotoxizität gegenüber malignen Zellen, welches auf eine Voraktivierung, gemessen an der Expression von Aktivierungsmarker CD69, schließen lässt. Die in vivo Untersuchungen zeigten, dass bei einer verabreichten T-Zell-Dosis unter 50 x 103/KG, nur milde GvHDs (Grad I/II) auftraten. Bis zu 60 x 106 NK-Zellen/KG wurden gut toleriert. Nebenwirkungen wie Fieber und Schüttelfrost waren transient und gingen einher mit erhöhten Zytokinspiegeln von inflammatorischen Zytokinen (IL-6, IL-8, IFN-γ) im Serum der Patienten. Ein engmaschiges Monitoring nach der NK-Zell-Applikation der IL-2 stimulierten NK-Zellen zeigte, dass die NK-Zellen in 5/6 Applikationen aus der peripheren Blutbahn abwanderten, einhergehend mit der Migration von Antigen-präsentierenden Zellen. Bei der Untersuchung des langfristigen Einflusses von adoptiver NK-Zell-Immuntherapie auf die Immunrekonstitution bei Patienten nach haploidenter SZT wurde vorausgehend eine Normwertstudie zu verschiedenen Leukozytensubpopulationen von 100 gesunden Kinder und Erwachsenen vorgenommen. Nach der Entwicklung eines stufenlosen, nicht-linearen Regressionsmodells konnte der Einfluss auf die Immunrekonstitution der Patienten nach SZT altersgerecht beurteilt werden. Weiterhin wurden Patientengruppen, die ebenfalls haploident transplantiert wurden, den NK-Zell-Studienpatienten gegenübergestellt. Eine signifikante Verbesserung durch die Gabe von NK-Zellen konnte nicht beobachtet werden. Zusammenfassend kann man sagen, dass die NK-Zellzahl innerhalb des ersten Monats nach SZT Normwerte erreichte, gefolgt von den zytotoxischen CD3+CD8+ T-Zellen 5-6 Monate nach haploidenter SZT, den THelfer Zellen und den B-Zellen nach über einem Jahr nach haploidenter SZT. Die allogene additive NK-Zell-Immuntherapie ist eine vielversprechende Therapieoption bei Patienten mit malignen Erkrankungen wie bspw. dem NB. Die NK-Zell-Aktivierung mit IL-2 bewies den Erhalt der Immunkompetenz. Dies war erkennbar an der gesteigerten zytotoxischen Funktionalität, der Zytokinproduktion und der Hochregulierung von zytotoxisch aktiven Rezeptoren. Eine verbesserte Immunrekonstitution kann durch das neue altersgerechte Lymphozyten-Norm-Modell besser beurteilt werden. Allerdings ist die Patientenanzahl und die Beobachtungszeit bisher zu gering, um in vivo ein verbessertes Überleben mit additiver NK-Zell-Immuntherapie wirklich abschätzen zu können

Pre-emptive allogeneic hematopoietic stem cell transplantation in ataxia telangiectasia

Ataxia telangiectasia (A-T) is a primary immunodeficiency with mutations in the gene encoding the A-T mutated (ATM) protein that interacts with immune, hematopoietic, and endocrine targets resulting in broad multi-systemic clinical manifestations with a devastating outcome. Apart from a progressive neurodegenerative disorder, A-T leads to significantly increased susceptibility to malignancies. It is a matter of discussion whether pre-emptive allogeneic hematopoietic stem cell transplantation (alloHSCT) using a reduced intensity conditioning regimen would be an option to restore immune-competence and prevent malignancy, as shown in animal models, because conventional treatment protocols of malignant diseases using radio- and/or chemotherapy have a high rate of therapy-related morbidity and mortality in these patients. We present the course of the disease, including immune reconstitution and neurological outcome following pre-emptive alloHSCT in a 4-year-old boy with A-T on a 6 year follow-up. Our manuscript provides a proof-of-concept of alloHSCT as an individual pre-emptive treatment strategy from which some A-T patients might benefit

Epstein-Barr virus–specific cytokine-induced killer cells for treatment of Epstein-Barr virus–related malignant lymphoma

Background: Prolonged immunosuppression or delayed T-cell recovery may favor Epstein-Barr virus (EBV) infection or reactivation after allogeneic hematopoietic stem cell transplantation (HSCT), which can lead to post-transplant lymphoproliferative disease (PTLD) and high-grade malignant B-cell lymphoma. Cytokine-induced killer (CIK) cells with dual specific anti-tumor and virus-specific cellular immunity may be applied in this context. Methods: CIK cells with EBV-specificity were generated from peripheral blood mononuclear cells (PBMCs), expanded in the presence of interferon-γ, anti-CD3, interleukin (IL)-2 and IL-15 and were pulsed twice with EBV consensus peptide pool. CIK cells with EBV-specificity and conventional CIK cells were phenotypically and functionally analyzed. Additionally, CIK cells with EBV-specificity were applied to a patient with EBV-related PTLD rapidly progressing to highly aggressive B-cell lymphoma on a compassionate use basis after approval and agreement by the regulatory authorities. Results: Pre-clinical analysis showed that generation of CIK cells with EBV-specificity was feasible. In vitro cytotoxicity analyses showed increased lysis of EBV-positive target cells, enhanced proliferative capacity and increased secretion of cytolytic and proinflammatory cytokines in the presence of EBV peptide-displaying target cells. In addition, 1 week after infusion of CIK cells with EBV-specificity, the patient's highly aggressive B-cell lymphoma persistently disappeared. CIK cells with EBV-specificity remained detectable for up to 32 days after infusion and infusion did not result in acute toxicity. Discussion: The transfer of both anti-cancer potential and T-cell memory against EBV infection provided by EBV peptide-induced CIK cells might be considered a therapy for EBV-related PTLD

Hematopoietic stem cell transplantation restores naïve t-cell populations in atm-deficient mice and in preemptively treated patients with ataxia-telangiectasia

Background: Ataxia-telangiectasia (A-T) is a multisystem disorder with progressive cerebellar ataxia, immunodeficiency, chromosomal instability, and increased cancer susceptibility. Cellular immunodeficiency is based on naïve CD4+ and CD8+ T-cell lymphopenia. Hematopoietic stem cell transplantation (HSCT) offers a potential to cure immunodeficiency and cancer due to restoration of the lymphopoietic system. The aim of this investigation was to analyze the effect of HSCT on naïve CD4+ as well as CD8+ T-cell numbers in A-T. Methods: We analyzed total numbers of peripheral naïve (CD45RA+CD62L+) and memory (CD45RO+CD62L−) CD4+ and CD8+ T-cells of 32 A-T patients. Naïve (CD62LhighCD44low) and memory (CD62LlowCD44high) T-cells were also measured in Atm-deficient mice before and after HSCT with GFP-expressing bone marrow derived hematopoietic stem cells. In addition, we analyzed T-cells in the peripheral blood of two A-T patients after HLA-identic allogeneic HSCT. Results: Like in humans, naïve CD4+ as well as naïve CD8+ lymphocytes were decreased in Atm-deficient mice. HSCT significantly inhibited thymic lymphomas and increased survival time in these animals. Donor cell chimerism increased up to more than 50% 6 months after HSCT accompanied by a significant increase of naïve CD4 and CD8 T-cell subpopulations, but not of memory T-cells. This finding was also identified in the blood of the A-T patients after HSCT. Conclusion: HSCT seems to be a feasible strategy to overcome immunodeficiency and might be a conceivable strategy to avoid T-cell driven cancer in A-T at higher risk for malignancy. Naïve CD4 and CD8 T-cells counts are suitable markers for monitoring immune reconstitution post-HSCT. However, risks and benefits of HSCT in A-T have to be properly weighted

Adoptive cellular immunotherapy for refractory childhood cancers: a single center experience

Prognosis of refractory childhood cancers despite multimodal treatment strategies remains poor. Here, we report a single center experience encountered in 18 patients with refractory solid malignancies treated with adoptive cellular immunotherapy (ACI) from haploidentical or matched donors following hematopoietic stem cell transplantation. While seven patients were in partial and six in complete remission (CR), five patients suffered from relapsed diseases at the time of ACI. 1.5-year probabilities of overall survival (OS) and progression-free survival (PFS) were 19.5% and 16.1% for all patients. Patients in CR showed estimated 1.5-year OS and PFS of 50.1% and 42.7%, respectively. CR was induced or rather sustained in ten children, with two still being alive 9.6 and 9.3 years after ACI. Naïve, central and effector memory T-cells correlated with responses. However, the majority of patients relapsed. Cumulative incidence of relapse was 79.8% at 1.5 years. Acute graft versus host disease (aGVHD) occurred in nine of 18 patients (50%) with aGVHD grade I–II observed in six (33%) and aGVHD grade III seen in three (17%) patients, manageable in all cases. Altogether, study results indicate that donor-derived ACI at its current state offers palliation but no clear curative benefit for refractory childhood cancers and warrants further improvement

The incidence and type of cancer in patients with ataxia-telangiectasia via a retrospective single-centre study

Ataxia-telangiectasia (A-T) is a hereditary immune system disorder with neurodegeneration. Its first neurologic symptoms include ataxic gait in early childhood, with slowly progressive cerebellar ataxia, oculomotor apraxia, oculocutaneous telangiectasia, and progressive muscle weakness. Neonatal screening for severe T-cell deficiency was recently found to diagnose A-T patients with a significantly reduced naïve T-cell pool. Our study includes 69 A-T patients between 8 January 2002 and 1 December 2019. Nineteen cases of cancer were diagnosed in 17 patients (25%), with a median overall survival [OS; 95% cumulative indcidence (CI)] of 26·9 years for the entire cohort. The 15-year OS of 82·5% (72–95%) was significantly decreased among A-T patients with malignancies, who had a median OS of 2·11 years, with a two-year-estimated OS of 50·7% (31–82%). Haematological malignancies were the major causes of death within the initial years of life with a 15 times increased risk for death [HR (95% CI): 6·9 (3·1–15.2), P < 0·001] upon malignancy diagnosis. Male patients with A-T are at a higher cancer risk than their female counterparts. This manuscript highlights the need for cancer surveillance and prevention, as well as optimal treatment in this cohort

The synergistic use of il-15 and il-21 for the generation of nk cells from cd3/cd19-depleted grafts improves their ex vivo expansion and cytotoxic potential against neuroblastoma: Perspective for optimized immunotherapy post haploidentical stem cell transplantation

Neuroblastoma (NB) is the most common solid extracranial tumor in childhood. Despite therapeutic progress, prognosis in high-risk NB is poor and innovative therapies are urgently needed. Therefore, we addressed the potential cytotoxic capacity of interleukin (IL)-activated natural killer (NK) cells compared to cytokine-induced killer (CIK) cells for the treatment of NB. NK cells were isolated from peripheral blood mononuclear cells (PBMCs) by indirect CD56-enrichment or CD3/CD19-depletion and expanded with different cytokine combinations, such as IL-2, IL-15, and/or IL-21 under feeder-cell free conditions. CIK cells were generated from PBMCs by ex vivo stimulation with interferon-γ, IL-2, OKT-3, and IL-15. Comparative analysis of expansion rate, purity, phenotype and cytotoxicity was performed. CD56-enriched NK cells showed a median expansion rate of 4.3-fold with up to 99% NK cell content. The cell product after CD3/CD19-depletion consisted of a median 43.5% NK cells that expanded significantly faster reaching also 99% of NK cell purity. After 10–12 days of expansion, both NK cell preparations showed a significantly higher median cytotoxic capacity against NB cells relative to CIK cells. Remarkably, these NK cells were also capable of efficiently killing NB spheroidal 3D culture in long-term cytotoxicity assays. Further optimization using a novel NK cell culture medium and a prolonged culturing procedure after CD3/CD19-depletion for up to 15 days enhanced the expansion rate up to 24.4-fold by maintaining the cytotoxic potential. Addition of an IL-21 boost prior to harvesting significantly increased the cytotoxicity. The final cell product consisted for the major part of CD16−, NCR-expressing, poly-functional NK cells with regard to cytokine production, CD107a degranulation and antitumor capacity. In summary, our study revealed that NK cells have a significantly higher cytotoxic potential to combat NB than CIK cell products, especially following the synergistic use of IL-15 and IL-21 for NK cell activation. Therefore, the use of IL-15+IL-21 expanded NK cells generated from CD3/CD19-depleted apheresis products seems to be highly promising as an immunotherapy in combination with haploidentical stem cell transplantation (SCT) for high-risk NB patients

Improving clinical manufacturing of IL-15 activated cytokine-induced killer (CIK) cells

Cytokine-induced killer (CIK) cells are an immunotherapeutic approach to combat relapse following allogeneic hematopoietic stem cell transplantation (HSCT) in acute leukemia or myelodysplastic syndrome (MDS) patients. Prompt and sequential administration of escalating cell doses improves the efficacy of CIK cell therapy without exacerbating graft vs. host disease (GVHD). This study addresses manufacturing-related issues and aimed to develop a time-, personal- and cost-saving good manufacturing process (GMP)-compliant protocol for the generation of ready-for-use therapeutic CIK cell doses starting from one unstimulated donor-derived peripheral blood (PB) or leukocytapheresis (LP) products. Culture medium with or without the addition of either AB serum, fresh frozen plasma (FFP) or platelet lysate (PL) was used for culture. Fresh and cryopreserved CIK cells were compared regarding expansion rate, viability, phenotype, and ability to inhibit leukemia growth. Cell numbers increased by a median factor of 10-fold in the presence of FFP, PL, or AB serum, whereas cultivation in FFP/PL-free or AB serum-free medium failed to promote adequate CIK cell proliferation (p < 0.01) needed to provide clinical doses of 1 × 106 T cells/kG, 5 × 106 T cells/kG, 1 × 107 T cells/kG, and 1 × 108 T cells/kG recipient body weight. CIK cells consisting of T cells, T- natural killer (T-NK) cells and a minor fraction of NK cells were not significantly modified by different medium supplements. Moreover, neither cytotoxic potential against leukemic THP-1 cells nor cell activation shown by CD25 expression were significantly influenced. Moreover, overnight and long-term cryopreservation had no significant effect on the composition of CIK cells, their phenotype or cytotoxic potential. A viability of almost 93% (range: 89–96) and 89.3% (range: 84–94) was obtained after freeze-thawing procedure and long-term storage, respectively, whereas viability was 96% (range: 90-97) in fresh CIK cells. Altogether, GMP-complaint CIK cell generation from an unstimulated donor-derived PB or LP products was feasible. Introducing FFP, which is easily accessible, into CIK cell cultures was time- and cost-saving without loss of viability and potency in a 10-12 day batch culture. The feasibility of cryopreservation enabled storage and delivery of sequential highly effective ready-for-use CIK cell doses and therefore reduced the number of manufacturing cycles

Pre-emptive immunotherapy for clearance of molecular disease in childhood acute lymphoblastic leukemia after transplantation

Monitoring of minimal residual disease (MRD) or chimerism may help guide pre-emptive immunotherapy (IT) with a view to preventing relapse in childhood acute lymphoblastic leukemia (ALL) after transplantation. Patients with ALL who consecutively underwent transplantation in Frankfurt/Main, Germany between January 1, 2005 and July 1, 2014 were included in this retrospective study. Chimerism monitoring was performed in all, and MRD assessment was performed in 58 of 89 patients. IT was guided in 19 of 24 patients with mixed chimerism (MC) and MRD and by MRD only in another 4 patients with complete chimerism (CC). The 3-year probabilities of event-free survival (EFS) were .69 ± .06 for the cohort without IT and .69 ± .10 for IT patients. Incidences of relapse (CIR) and treatment-related mortality (CITRM) were equally distributed between both cohorts (without IT: 3-year CIR, .21 ± .05, 3-year CITRM, .10 ± .04; IT patients: 3-year CIR, .18 ± .09, 3-year CITRM .13 ± .07). Accordingly, 3-year EFS and 3-year CIR were similar in CC and MC patients with IT, whereas MC patients without IT experienced relapse. IT was neither associated with an enhanced immune recovery nor an increased risk for acute graft-versus-host disease. Relapse prevention by IT in patients at risk may lead to the same favorable outcome as found in CC and MRD-negative-patients. This underlines the importance of excellent MRD and chimerism monitoring after transplantation as the basis for IT to improve survival in childhood ALL

Development of three different NK cell subpopulations during immune reconstitution after pediatric allogeneic hematopoietic stem cell transplantation: prognostic markers in GvHD and viral infections

Natural killer (NK) cells play an important role following allogeneic hematopoietic stem cell transplantation (HSCT) exerting graft-versus-leukemia/tumor effect and mediating pathogen-specific immunity. Although NK cells are the first donor-derived lymphocytes reconstituting post-HSCT, their distribution of CD56++CD16− (CD56bright), CD56++CD16+ (CD56intermediate=int), and CD56+CD16++ (CD56dim) NK cells is explicitly divergent from healthy adults, but to some extent comparable to the NK cell development in early childhood. The proportion of CD56bright/CD56int/CD56dim changed from 15/8/78% in early childhood to 6/4/90% in adults, respectively. Within this study, we first compared the NK cell reconstitution post-HSCT to reference values of NK cell subpopulations of healthy children. Afterward, we investigated the reconstitution of NK cell subpopulations post-HSCT in correlation to acute graft versus host disease (aGvHD) and chronic graft versus host disease (cGvHD) as well as to viral infections. Interestingly, after a HSCT follow-up phase of 12 months, the distribution of NK cell subpopulations largely matched the 50th percentile of the reference range for healthy individuals. Patients suffering from aGvHD and cGvHD showed a delayed reconstitution of NK cells. Remarkably, within the first 2 months post-HSCT, patients suffering from aGvHD had significantly lower levels of CD56bright NK cells compared to patients without viral infection or without graft versus host disease (GvHD). Therefore, the amount of CD56bright NK cells might serve as an early prognostic factor for GvHD development. Furthermore, a prolonged and elevated peak in CD56int NK cells seemed to be characteristic for the chronification of GvHD. In context of viral infection, a slightly lower CD56 and CD16 receptor expression followed by a considerable reduction in the absolute CD56dim NK cell numbers combined with reoccurrence of CD56int NK cells was observed. Our results suggest that a precise analysis of the reconstitution of NK cell subpopulations post-HSCT might indicate the occurrence of undesired events post-HSCT such as severe aGvHD.value