Bortezomib promotes the TRAIL-mediated killing of resistant rhabdomyosarcoma by ErbB2/Her2-targeted CAR-NK-92 cells via DR5 upregulation.

Treatment resistance and immune escape are hallmarks of metastatic rhabdomyosarcoma (RMS), underscoring the urgent medical need for therapeutic agents against this disease entity as a key challenge in pediatric oncology. Chimeric antigen receptor (CAR)-based immunotherapies, such as the ErbB2 (Her2)-CAR-engineered natural killer (NK) cell line NK-92/5.28.z, provide antitumor cytotoxicity primarily through CAR-mediated cytotoxic granule release and thereafter-even in cases with low surface antigen expression or tumor escape-by triggering intrinsic NK cell-mediated apoptosis induction via additional ligand/receptors. In this study, we showed that bortezomib increased susceptibility toward apoptosis in clinically relevant RMS cell lines RH30 and RH41, and patient-derived RMS tumor organoid RMS335, by upregulation of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor DR5 in these metastatic, relapsed/refractory (r/r) RMS tumors. Subsequent administration of NK-92/5.28.z cells significantly enhanced antitumor activity in vitro. Applying recombinant TRAIL instead of NK-92/5.28.z cells confirmed that the synergistic antitumor effects of the combination treatment were mediated via TRAIL. Western blot analyses indicated that the combination treatment with bortezomib and NK-92/5.28.z cells increased apoptosis by interacting with the nuclear factor κB, JNK, and caspase pathways. Overall, bortezomib pretreatment can sensitize r/r RMS tumors to CAR- and, by upregulating DR5, TRAIL-mediated cytotoxicity of NK-92/5.28.z cells.

Trends and Differences in Status Epilepticus Treatment of Children and Adults Over 10 Years: A Comparative Study of Medical Records (2012-2021) from a University Hospital in Germany.

BACKGROUND AND OBJECTIVES: Over the last decade, significant advancements have been made in status epilepticus (SE) management, influenced by landmark trials such as ESETT and RAMPART. The objectives of this study were to explore the evolution of drug treatments for patients with SE, to investigate its association with outcomes and mortality, and to evaluate differences in treatment patterns between adults and children for a potential shift in medication trends due to the above mentioned trials. METHODS: The medical records of patients with SE treated at University Hospital Frankfurt between 2012 and 2021 were evaluated for medication trends and outcomes. Children and adults were analyzed separately and jointly. RESULTS: This study included 1151 SE episodes in 1021 patients (mean age = 53.3 ± 28.3 years; 52.5 % female [n = 533]). The overall percentage of patients with SE treated prehospital was stable over the last decade. More than half (53.6 %) of children were treated prehospital, compared with less than one-third (26.7 %) of adults. Prehospital midazolam use increased over time, while diazepam use decreased. Lorazepam was the most commonly used benzodiazepine in hospitals in 2012-2013, used in 40.8 % of all episodes. However, its use declined to 27.2 % in 2020-2021, while midazolam use increased to 44.0 %. While the use of older antiseizure medications (ASMs) such as phenobarbital (p = 0.02), phenytoin (p < 0.001), and valproate (p < 0.001) decreased, the use of newer ASMs such as levetiracetam and lacosamide significantly increased (p < 0.001). Propofol and continuous midazolam infusion remained the most used third-line therapy drugs. Overall mortality was 16.5 % at discharge and 18.9 % at 30 days. Mortality rates did not change between 2012 and 2021. CONCLUSION: Midazolam has become the preferred benzodiazepine in pre- and in-hospital settings, both in children and adults. The same applies to the increased use of levetiracetam and lacosamide over time in children and adults, while phenobarbital, phenytoin, and valproate use decreased. Continuous midazolam infusion and propofol remain the most frequently used anesthetic drugs. Mortality and outcome remain stable despite changes in medication patterns.

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

Introduction: Metastatic 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. Methods: Here, 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. Results: Our 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. Discussion: These findings encourage further development of NK-92/5.28.z cells as off-the-shelf immunotherapy for the treatment of metastatic RMS.

Pediatric ICU Admissions After Adolescent Suicide Attempts During the Pandemic.

BACKGROUND AND OBJECTIVES: The worldwide severe acute respiratory syndrome coronavirus 2 pandemic challenges adolescents' mental health. In this study, we aim to compare the number of pediatric ICU (PICU) admissions after suicide attempts during the first German lockdown and one year later during a second, prolonged lockdown with prepandemic years. METHODS: A retrospective multicenter study was conducted among 27 German PICUs. Cases <18 years admitted to the PICU because of accidents or injuries between March 16 and May 31 of 2017 to 2021 were identified based on International Classification of Diseases, 10th Revision codes (German modification) and patient data entered into a database. This study is a subset analysis on suicide attempts in adolescents aged 12 to 17.9 years. The Federal Statistics Office was queried for data on fatal suicides, which were available only for 2020 in adolescents aged 10 to 17.9 years. RESULTS: Total admissions and suicide attempts declined during the first lockdown in 2020 (standardized morbidity ratio 0.74 (95% confidence interval; 0.58-0.92) and 0.69 (0.43-1.04), respectively) and increased in 2021 (standardized morbidity ratio 2.14 [1.86-2.45] and 2.84 [2.29-3.49], respectively). Fatal suicide rates remained stable between 2017 to 2019 and 2020 (1.57 vs 1.48 per 100 000 adolescent years) with monthly numbers showing no clear trend during the course of 2020. CONCLUSIONS: This study shows a strong increase in serious suicide attempts among adolescents during the course of the pandemic in Germany. More research is needed to understand the relation between pandemic prevention measures and suicidal ideation to help implement mental health support for adolescents.

Impact of the First COVID Lockdown on Accident- and Injury-Related Pediatric Intensive Care Admissions in Germany-A Multicenter Study.

Children's and adolescents' lives drastically changed during COVID lockdowns worldwide. To compare accident- and injury-related admissions to pediatric intensive care units (PICU) during the first German COVID lockdown with previous years, we conducted a retrospective multicenter study among 37 PICUs (21.5% of German PICU capacities). A total of 1444 admissions after accidents or injuries during the first lockdown period and matched periods of 2017-2019 were reported and standardized morbidity ratios (SMR) were calculated. Total PICU admissions due to accidents/injuries declined from an average of 366 to 346 (SMR 0.95 (CI 0.85-1.05)). Admissions with trauma increased from 196 to 212 (1.07 (0.93-1.23). Traffic accidents and school/kindergarten accidents decreased (0.77 (0.57-1.02 and 0.26 (0.05-0.75)), whereas household and leisure accidents increased (1.33 (1.06-1.66) and 1.34 (1.06-1.67)). Less neurosurgeries and more visceral surgeries were performed (0.69 (0.38-1.16) and 2.09 (1.19-3.39)). Non-accidental non-suicidal injuries declined (0.73 (0.42-1.17)). Suicide attempts increased in adolescent boys (1.38 (0.51-3.02)), but decreased in adolescent girls (0.56 (0.32-0.79)). In summary, changed trauma mechanisms entailed different surgeries compared to previous years. We found no evidence for an increase in child abuse cases requiring intensive care. The increase in suicide attempts among boys demands investigation.

Retargeting of NK-92 Cells against High-Risk Rhabdomyosarcomas by Means of an ERBB2 (HER2/Neu)-Specific Chimeric Antigen Receptor.

The dismal prognosis of pediatric and young adult patients with high-risk rhabdomyosarcoma (RMS) underscores the need for novel treatment options for this patient group. In previous studies, the tumor-associated surface antigen ERBB2 (HER2/neu) was identified as targetable in high-risk RMS. As a proof of concept, in this study, a novel treatment approach against RMS tumors using a genetically modified natural killer (NK)-92 cell line (NK-92/5.28.z) as an off-the-shelf ERBB2-chimeric antigen receptor (CAR)-engineered cell product was preclinically explored. In cytotoxicity assays, NK-92/5.28.z cells specifically recognized and efficiently eliminated RMS cell suspensions, tumor cell monolayers, and 3D tumor spheroids via the ERBB2-CAR even at effector-to-target ratios as low as 1:1. In contrast to unmodified parental NK-92 cells, which failed to lyse RMS cells, NK-92/5.28.z cells proliferated and became further activated through contact with ERBB2-positive tumor cells. Furthermore, high amounts of effector molecules, such as proinflammatory and antitumoral cytokines, were found in cocultures of NK-92/5.28.z cells with tumor cells. Taken together, our data suggest the enormous potential of this approach for improving the immunotherapy of treatment-resistant tumors, revealing the dual role of NK-92/5.28.z cells as CAR-targeted killers and modulators of endogenous adaptive immunity even in the inhibitory tumor microenvironment of high-risk RMS.

Depletion of CD45RA+ T cells: Advantages and disadvantages of different purification methods.

BACKGROUND: Recently, new advances were made regarding the depletion of CD45RA+ naïve T cells from haploidentical grafts as they are suspected to be the most alloreactive. METHODS: Within this project we investigated CD45RA-depletion from G-CSF mobilized PBSC by two different purification strategies according to GMP, specifically direct depletion of CD45RA+ cells (one-step approach), or CD34-positive selection followed by CD45RA-depletion (two-step approach). RESULTS: With log -3.9 and - 3.8 the depletion quality of CD45RA+ T cells was equally for both approaches together with a close to complete CD19+ B cell depletion. However, due to a high expression of CD45RA the majority of NK cells were lost within both CD45RA depletion strategies. Stem cell recovery after one-step CD45RA-depletion was at median 52.0% (range: 49.7-67.2%), which was comparable to previously published recovery data received from direct CD34 positive selection. Memory T cell recovery including CD4+ and CD8+ memory T cell subsets was statistically not differing between both purification approaches. The recovery of CD4+ and CD8+ T cells was as well similar, but overall a higher amount of cytotoxic than T-helper cells were lost as indicated by an increase of the CD4/CD8 ratio. CONCLUSIONS: CD45RA-depletion from G-CSF mobilized PBSC is feasible as one- and two-step approach and results in sufficient reduction of CD45RA+ T cells as well as B cells, but also to a co-depletion of NK cells. However, by gaining two independent cell products, the two-step approach enables the highest clinical flexibility in regard to individual graft composition with precise dosage of stem cells and T cells.

ERBB2-CAR-Engineered Cytokine-Induced Killer Cells Exhibit Both CAR-Mediated and Innate Immunity Against High-Risk Rhabdomyosarcoma.

High-risk rhabdomyosarcoma (RMS) occurring in childhood to young adulthood is associated with a poor prognosis; especially children above the age of 10 with advanced stage alveolar RMS still succumb to the disease within a median of 2 years. The advent of chimeric antigen receptor (CAR)-engineered T cells marked significant progress in the treatment of refractory B cell malignancies, but experience for solid tumors has proven challenging. We speculate that this is at least in part due to the poor quality of the patient's own T cells and therefore propose using CAR-modified cytokine-induced killer (CIK) cells as effector cells. CIK cells are a heterogeneous population of polyclonal T cells that acquire phenotypic and cytotoxic properties of natural killer (NK) cells through the cultivation process, becoming so-called T-NK cells. CIK cells can be genetically modified to express CARs. They are minimally alloreactive and can therefore be acquired from haploidentical first-degree relatives. Here, we explored the potential of ERBB2-CAR-modified random-donor CIK cells as a treatment for RMS in xenotolerant mice bearing disseminated high-risk RMS tumors. In otherwise untreated mice, RMS tumors engrafted 13-35 days after intravenous tumor cell injection, as shown by in vivo bioluminescence imaging, immunohistochemistry, and polymerase chain reaction for human gDNA, and mice died shortly thereafter (median/range: 62/56-66 days, n = 5). Wild-type (WT) CIK cells given at an early stage delayed and eliminated RMS engraftment in 4 of 6 (67%) mice, while ERBB2-CAR CIK cells inhibited initial tumor load in 8 of 8 (100%) mice. WT CIK cells were detectable but not as active as CAR CIK cells at distant tumor sites. CIK cell therapies during advanced RMS delayed but did not inhibit tumor progression compared to untreated controls. ERBB2-CAR CIK cell therapy also supported innate immunity as evidenced by selective accumulation of NK and T-NK cell subpopulations in disseminated RMS tumors, which was not observed for WT CIK cells. Our data underscore the power of heterogenous immune cell populations (T, NK, and T-NK cells) to control solid tumors, which can be further enhanced with CARs, suggesting ERBB2-CAR CIK cells as a potential treatment for high-risk RMS.

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.

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.

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.

Clearance of Hematologic Malignancies by Allogeneic Cytokine-Induced Killer Cell or Donor Lymphocyte Infusions.

Well-established donor lymphocyte infusion (DLI) and novel cytokine-induced killer (CIK) cell therapy for the treatment of relapsing hematologic malignancies after allogeneic hematopoietic stem cell transplantation (HSCT) were compared with respect to feasibility, safety, and efficacy. Altogether, a total of 221 infusions were given to 91 patients (DLI, n = 55; CIK, n = 36). T cell recovery was significantly improved after CIK cell therapy (P < .0001). Although patients with CIK cell treatment showed a significantly worse prognosis at the time of HSCT (risk score, 1.7 versus 2.1; P < .0001), DLI and CIK cell therapy induced complete remission (CR) in 29% and 53% patients, respectively, whereas relapse occurred in 71% and 47%. In both groups, all patients with overt hematologic relapse at the time of immunotherapy (DLI, n = 11; CIK, n = 8) succumbed to their disease, while 36% and 68% patients with DLI or CIK cell therapy applied due to molecular relapse or active disease at the time of transplantation achieved CR. The 6-month overall survival rate in the latter patients was 57% and 77%, respectively, with a median follow-up of 27.9 months (range, .9 to 149.2 months). The 6-month cumulative incidence of relapse was 55% and 22% in patients who received DLI and CIK cell therapy, respectively (P = .012). Acute graft-versus-host disease developed in 35% of the patients who received DLI and in 25% of those who received CIK. No transfusion-related deaths occurred. These data, while underscoring the therapeutic value of conventional DLI, suggest the improved safety and to a certain extent efficacy of CIK cell therapy for patients at high risk for post-transplantation relapse of various hematologic malignancies.

AlloHSCT in paediatric ALL and AML in complete remission: improvement over time impacted by accreditation?

Allogeneic hematopoietic stem cell transplantation (alloHSCT) has become a well-established treatment option for many patients suffering from malignant and non-malignant diseases. In the past decade, high-resolution HLA-typing, remission surveillance, pre-emptive immune intervention, and standardisation in supportive care measures have substantially improved transplant outcomes. This retrospective study evaluated transplant procedures in 162 paediatric patients with acute lymphoblastic leukaemia (n = 124) or acute myeloid leukaemia (n = 38) who received their first alloHSCT in our institution over an 11-year period. We observed a significant reduction in risk of non-relapse mortality (NRM) over time (HR = 0.34, 95% CI 0.12-0.98; P = 0.05), the 4-year NRM estimate decreased from 20% in 2005-2008 to 7% in 2012-2016 (P = 0.02) and an increase in survival after relapse. There was no significant difference in patients who received a graft from a sibling, haplo, or an unrelated donor with regard to their overall survival (P = 0.45), event-free survival (P = 0.61), and non-relapse mortality (P = 0.19). Our data suggest that a specific transplant infrastructure with a highly experienced team in an accredited transplant centre likely contributes to better transplant outcomes for acute leukaemia patients in complete remission regardless of donor type.

Generation and characterization of ErbB2-CAR-engineered cytokine-induced killer cells for the treatment of high-risk soft tissue sarcoma in children.

Pediatric patients with recurrent, refractory or advanced soft tissue sarcoma (STS) who are simultaneously showing signs of cumulative treatment toxicity are in need of novel therapies. In this preclinical analysis, we identified ErbB2 as a targetable antigen on STS cells and used cytokine-induced killer (CIK) cells transduced with the lentiviral 2nd-generation chimeric antigen receptor (CAR) vector pS-5.28.z-IEW to target ErbB2-positive tumors. Solely CIK cell subsets with the CD3+ T cell phenotype showed up to 85% cell surface expression of the respective CAR. A comparison of wildtype (WT), mock-vector and ErbB2-CAR-CIK cells showed, that engineered cells exhibited diminished in vitro expansion, retained WT CIK cell phenotype with higher percentages of differentiated effector memory/effector cells. Activating natural killer (NK) cell receptor NKG2D-restricted target cell recognition and killing of WT and ErbB2-CAR-CIK cells was maintained against ErbB2-negative tumors, while ErbB2-CAR-CIK cells demonstrated significantly increased cytotoxicity against ErbB2-positive targets, including primary tumors. ErbB2-CAR- but not WT CIK cells proliferated, infiltrated and efficiently lysed tumor cell monolayers as well as 3D tumor spheroids. Here, we demonstrate a potential cell therapeutic approach using ErbB2-CAR-CIK cells for the recognition and elimination of tumor cells expressing ErbB2, which we identified as a targetable antigen on high-risk STS cells.

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.

Cytomegalovirus-specific cytokine-induced killer cells: concurrent targeting of leukemia and cytomegalovirus.

BACKGROUND AIMS: Human cytomegalovirus (CMV) infection and reactivation is a leading complication of allogeneic hematopoietic stem cell transplantation (HSCT). In addition to drug treatment, the adoptive transfer of virus-specific T cells to restore cellular immunity has become a standard therapy after allogeneic HSCT. We recently demonstrated potent anti-leukemic activity of interleukin (IL)-15-activated cytokine-induced killer (CIK) cells. With the use of the same expansion protocol, we asked whether concurrent CMV antigen-pulsing might generate CIK cells with anti-leukemic and anti-CMV activity. METHODS: CIK cells expanded in the presence of interferon-γ, IL-2, IL-15 and anti-CD3 antibody were pulsed once with CMV(pp65) peptide pool. CMV-specific CIK (CIK(pp65)) and conventional CIK cells were phenotypically and functionally characterized according to their cytokine secretion pattern, degranulation capacity and T-cell receptor (TCR)-mediated and NKG2D-mediated cytotoxicity. RESULTS: We demonstrated that among CIK cells generated from CMV-seropositive donors, a single stimulation with CMV(pp65) protein co-expanded cytotoxic CMV-specific cells without sacrificing anti-tumor reactivity. Cells generated in this fashion lysed CMV(pp65)-loaded target cells and CMV-infected fibroblasts but also leukemic cells. Meanwhile, the alloreactive potential of CIK(pp65) cells remained low. Interestingly, CMV reactivity was TCR-mediated and CMV-specific cells could be found in CD3(+)CD8(+)CD56(+/-) cytotoxic T-cell subpopulations. CONCLUSIONS: We provide an efficient method to generate CIK(pp65) cells that may represent a useful cell therapy approach for preemptive immunotherapy in patients who have both an apparent risk of CMV and impending leukemic relapse after allogeneic stem cell transplantation.

Immunomagnetic selection or irradiation eliminates alloreactive cells but also reduces anti-tumor potential of cytokine-induced killer cells: implications for unmanipulated cytokine-induced killer cell infusion.

BACKGROUND AIMS: Cytokine-induced killer (CIK) cells may offer a novel therapeutic approach for patients with malignancies relapsing after allogeneic stem cell transplantation. Although CIK cells display negligible alloreactivity and cause minimal graft versus-host-disease (GVHD), high CIK cell doses required during relapse may pose a risk for severe GVHD, specifically in the mismatched or haploidentical transplantation setting. Manipulation of CIK cells may reduce risk for GVHD without affecting the anti-tumor potential. METHODS: In this pre-clinical study, we provide a detailed functional comparison of conventional and irradiated, CD56-enriched or T-cell receptor α/ß-depleted CIK cells. RESULTS: In vitro analysis showed retained anti-leukemic and anti-tumor potential after CIK cell manipulation. Even being sequentially infused into immunodeficient mice grafted with malignant cells, cytotoxic effects were fewest after irradiation but were improved by CD56 enrichment and were best with conventional CIK cells. Hence, considering the proliferative capacity of inoculated malignancies and effector cells, a single dose of conventional CIK cells resulted in prolonged disease-free survival and elimination of rhabdomyosarcoma cells, whereas sequential infusions were needed to achieve comparable results in leukemia-bearing mice. However, this mouse model has limitations: highly effective conventional CIK cells demonstrated both limited xenogenic GVHD and low alloreactive potential in vitro. CONCLUSIONS: Our study revealed that conventional CIK cells demonstrate no significant alloreactive potential but provide the strongest anti-tumor efficacy compared with manipulated CIK cells. Conventional CIK cells may therefore be tested in high numbers and short-term intervals in patients with impending relapse even after mismatched transplantation.