Background: Acute myeloid leukemia (AML) is a heterogenous malignancy and efficient treatment strategies for high-risk patients are still lacking. To facilitate preclinical and translational leukemia research, e.g. to test novel therapeutics in vivo, we generate patient-derived xenograft (PDX) samples. We had transplanted more than 100 primary adult AML samples into immune-compromised NSG mice. Furthermore, we received PDX samples primografted in other labs of pediatric and adult patients. However, only around 20% of primary samples (i.e. “high score samples”) show a robust and reliable serial engraftment and can be genetically modified, facilitating repetitive, reproducible and convenient in vivo experiments. Aims: (i) Identifying factors influencing reliable serial engraftment; (ii) Characterizing high score samples functionally and genetically; (iii) Performing in vivo (therapy) trials. Methods: We generated 23 high score AML PDX samples characterized by (i) a serial, fast, reliable and systemic engraftment, and (ii) the capacity to be genetically engineered. These samples were characterized by DNA panel sequencing (seq), whole exome seq, low coverage whole genome seq, RNA seq, methylation profiling, karyotyping, and immunophenotyping. Furthermore, samples were transplanted repetitively, and capacity for serial engraftment was analyzed. Last, samples were used for diverse in vivo trials, e.g. long-term chemotherapy trials. Results: High score samples originated mainly from relapsed AML cases (18/23, p<0.05). Overall survival (OS) of patients whose cells yielded high score samples was dramatically lower compared to OS of patients whose cells did not engraft or meet the criteria of high score samples (p<0.01). Analysis of AML-related mutations, gene fusions, translocations, copy number alterations and immunophenotype revealed that high score AML PDX samples resembled primary patient cells of both, initial diagnosis and relapsed disease, and - in contrast to established AML cell lines - covered many different genetic and genomic alterations. Within the WHO classification of 2022, most samples belonged to one of the three groups “AML with NPM1 mutation” (n=8), “AML with KMT2A rearrangement” (n=7), or “AML, myelodysplasia-related” (n=6). High score samples were serially transplanted into recipient mice, with up to fifteen repetitive transplantations. Importantly, we did not lose a sample in serial re-transplantations, indicating that leukemia initiating cells proliferated within the murine niche for many months (median 412 days). On the contrary, under in vitro conditions, cells could be kept for some days to weeks, but ultimately died. With every engraftment, PDX cells can be amplified by a factor 30-fold or higher, depending on the sample, allowing the production of indefinite numbers of PDX cells. Engraftment capacity and engraftment time were influenced by several factors; thawed PDX cells and injection of cell numbers below 500,000 cells had a reduced engraftment rate and a longer passaging time compared to freshly isolated cells or higher cells numbers. Serial re-passaging or expression of transgenes did, however, not influence engraftment. While murine gender influenced engraftment capacity of some samples, with male mice being less supportive, mouse age did not, and even older mice with more than 20 weeks of age allowed reliable engraftment. High score AML PDX samples were further used in preclinical in vivo therapy trials. Stable luciferase expression allowed sensitive disease monitoring, facilitating real-time analysis of drug effects and of growth kinetics before and after treatment. Bioluminescence imaging revealed that azacitidine, in contrast to cytarabine, had a delayed effect on tumor burden. Both drugs, however, showed a sustained effect on the cells even after stop of long-term therapy, indicated by a reduced growth rate after treatment. Conclusion and Outlook: High score AML PDX models represent highly aggressive primary patients' cells. By serial engraftment and genetic engineering, we can produce indefinite numbers of these cells, which represent a valuable and unique tool for sophisticated molecular and functional studies. Our well characterized AML PDX cohort allows repetitive and reproducible preclinical trials, and luciferase expression facilitates reliable monitoring of long-term therapy trials.

Preclinical cancer research requires robust model systems, especially for poor prognosis entities like acute myeloid leukemia (AML), a highly aggressive blood cancer. Here, primary tumor cells from 137 AML patients of all age groups were transplanted into immune compromised mice to generate patient-derived xenografts (PDX). From these, 23 models enable robust, virtually endless serial re-transplantation and are amenable to lentiviral genetic engineering (*PDX AML models). These models primarily originate from patients with highly aggressive, relapsed disease. Comprehensive genomic, transcriptomic, and epigenomic analyses confirmed that they replicate primary AML biology more faithfully than conventional cell lines. Notably, *PDX AML models include AML subgroups that are underrepresented or absent in existing model systems, such as cytogenetically normal or IDH1/2 -mutant AML. They withstand freeze-thaw cycles, making them suitable for broad distribution and reproducibility across research institutions. Luciferase-based in vivo imaging enables real-time monitoring of tumor progression and treatment responses in preclinical trials. Surprisingly, long-term treatment, including repeated cytarabine therapy over a period of one year, showed a gradual reduction in leukemia cell proliferation, which decreased continuously after each treatment block. Collectively, our *PDX models represent a robust, versatile, and relevant platform that holds great promise to accelerate translational research for the benefit of cancer patients. ![Figure][1] Key Points ### Competing Interest Statement TH: Travel support: Jazz Pharmaceuticals; Participation on Advisory Board: Servier and Jazz Pharmaceuticals; honoraria for speakers: Astellas Pharma. All other authors declare no competing interest. European Research Council, https://ror.org/0472cxd90, 681524 Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, 537701335, SFB 1709/1 2025 - 533056198 German Cancer Aid, https://ror.org/01wxdd722, Mildred Scheel Professorship Deutsche José Carreras Leukämie-Stiftung, https://ror.org/00826gz80, DJCLS 15 R/2021, DJCLS 02 R/2023 Bettina Bräu Stiftung Dr. Helmut Legerlotz Stiftung [1]: pending:yes

Chimeric antigen receptor (CAR) T-cell therapy directed to CD19 and B-cell maturation antigen has revolutionized treatment of B-cell leukemia and lymphoma, and multiple myeloma. However, identifying suitable targets for acute myeloid leukemia (AML) remains challenging due to concurrent expression of potential target antigens on normal hematopoietic stem cells or tissues. As the stress-induced B7H6 molecule is rarely found on normal tissues but expressed on many cancers including AML and melanoma, the NKp30-ligand B7H6 emerges as a promising target for NKp30-based CAR T therapy for these tumors. In this study, we report a comprehensive B7H6 expression analysis on primary AML and melanoma as well as on different tumor cell-lines examined by RT-qPCR and flow cytometry, and efficient anti-tumor reactivity of NKp30-CAR T cells to AML and melanoma. To overcome limitations of autologous CAR T-cell fitness-dependent efficacy and patient-tailored production, we generated CRISPR/Cas9-mediated TCR-knockout (TCRKO) NKp30-CAR T cells as an off-the-shelf approach for CAR T therapy. Functional studies comparing NKp30-CD28 CAR or NKp30-CD137 CAR TCR+ and TCRKO T lymphocytes revealed superior anti-tumoral immunity of NKp30-CD28 CAR TCRKO T cells to AML and melanoma cell lines in vitro, and effective control of tumor burden in an NSG melanoma-xenograft mouse model. In conclusion, these findings highlight the therapeutic potential of NKp30 CAR TCRKO T cells for adoptive T-cell therapy to B7H6-expressing cancers, including melanoma and AML.

PIMS-TS presents with features considered to pose a risk for the development of unfavourable long-term mental health outcomes which haven’t been prospectively investigated. These include neurological symptoms (headache, confusion, and delirium), systemic inflammation, and critical illness requiring PICU support. Therefore, we set out to investigate mental health at 6-month follow-up in patients recruited into Swissped-RECOVERY trial. This is a pre-planned analysis of the Swissped-RECOVERY trial (clinicaltrial.gov, NCT 04826588) investigating intravenous immunoglobulins versus intravenous methylprednisolone in patients with PIMS-TS. Mental health was assessed 6 months after hospital discharge using the Strength and Difficulties Questionnaire (SDQ), leading to a score concerning mental well-being (total score scale 0–40 points, subdomains 0–10 points). At population level it is expected that 80 https://clinicaltrials.gov/study/NCT04826588 .

Data on COVID-19 vaccine acceptability among parents of children with multisystem inflammatory syndrome (MIS-C) are limited. In this cohort of children with MIS-C, enrolled in the Swissped RECOVERY trial (NCT04826588), comparing intravenous immunoglobulins or methylprednisolone, who, in accordance with Swiss guidelines, were recommended for SARS-CoV-2 vaccination, 65% (73/112) of parents reported being vaccinated against SARS-CoV-2 before the MIS-C, while 70% were vaccinated after the MIS-C episode of their child. None of the children were vaccinated before the occurrence of the MIS-C, and only 9% (5/56) received the COVID-19 vaccine after the MIS-C. The predominant barriers to COVID-19 vaccination were concerns over potential side effects and insufficient support from their doctors. This emphasizes the crucial role of health care providers in promoting COVID-19 vaccination among children.

TIGIT is an alternative checkpoint receptor (CR) whose inhibition promotes Graft-versus-Leukemia effects of NK cells. Given the significant immune-permissiveness of NK cells circulating in acute myeloid leukemia (AML) patients, we asked whether adoptive transfer of activated NK cells would benefit from additional TIGIT-blockade. Hence, we characterized cytokine-induced memory-like (CIML)-NK cells and NK cell lines for the expression of inhibitory CRs. In addition, we analyzed the transcription of CR ligands in AML patients (CCLE and Beat AML 2.0 cohort) in silico and evaluated the efficacy of CR blockade using in vitro cytotoxicity assays, CD69, CD107a and IFN-γ expression. Alternative but not classical CRs were abundantly expressed on healthy donor NK cells and even further upregulated on CIML-NK cells. In line with our finding that CD155, one important TIGIT-ligand, is reliably expressed on AMLs, we show improved killing of CD155+-AML blasts by NK-92 but interestingly not CIML-NK cells in the presence of TIGIT-blockade. Additionally, our in silico data (n = 671) show that poor prognosis AML patients rather displayed a CD86low CD112/CD155high phenotype, whereas patients with a better outcome rather exhibited a CD86high CD112/CD155low phenotype. Collectively, our data evidence that the complex CR ligand expression profile on AML blasts may be one explanation for the intrinsic NK cell exhaustion observed in AML patients which might be overcome with adoptive NK-92 transfer in combination with TIGIT-blockade.

Recent studies indicate that under certain conditions such as viral infection or exposure to pro-inflammatory cytokines, NK cells may acquire features of adaptive immune cells. In this context, various forms of adaptive NK cells have been described, i.e. "liver-resident" memory-like NK cells, cytomegalovirus (CMV)-induced memory NK cells and interleukin (IL)12/15/18 cytokine-induced memory-like (CIML)-NK cells. We recently provided evidence that upon a 7-day co-culture with irradiated leukemia specimens NK cells can exhibit a memory-like phenotype with substantial anti-leukemic functionality. Here, we propose an antibody panel that allows the identification of subtle changes in the activation status and maturation during memory cell conversion of these so-called tumor-induced memory-like (TIML)-NK cells but also the comparison of those with other forms of memory NK cells. As tremendous efforts are currently undertaken to evaluate the clinical benefit of adoptive cell transfer of various forms of NK cells, we here delineate the process of our panel design in detail to provide future researchers with the means to optimize the flow cytometric analysis of various forms of memory NK cells within their clinical trial protocols.

Background Previous findings from the Swissped RECOVERY trial showed that patients with Pediatric Inflammatory Multisystem Syndrome-Temporally Associated with SARS-CoV-2 (PIMS-TS) who were randomly assigned to intravenous immunoglobulins or methylprednisolone have a comparable length of hospital stay. Here, we report the 6-month follow-up outcomes of cardiac pathologies and normalisation of clinical or laboratory signs of inflammation from this study population. Methods This pre-planned follow-up of patients with PIMS-TS included the Swissped RECOVERY Trial reports on the 6-month outcomes of the cohort after randomisation, with a focus on cardiac, haematological, and biochemical findings. The trial was an investigator-initiated randomised multicentre open-label two-arm trial in children and adolescents hospitalised with PIMS-TS at ten hospitals in Switzerland. Cardiological assessments and laboratory analyses were prospectively collected in the intention-to-treat analysis on pre-defined intervals after hospital discharge. Differences between randomised arms were investigated using Chi-square test for categorical and Wilcoxon test for continuous variables. The trial is registered with the Swiss National Clinical Trials Portal (SNCTP000004720) and ClinicalTrials.gov (NCT04826588). Findings Between May 21, 2021 and April 15, 2022, 75 patients with a median age of 9.1 years (IQR 6.2-12.2) were included in the intention-to-treat population (37 in the methylprednisolone group and 38 in the intravenous immunoglobulin group). During follow-up, the incidence of abnormal left ventricular systolic function, coronary artery aneurysms (CAA), and other signs of inflammation were comparable in both groups. However, we detected cardiac abnormalities with low incidence and a mild degree grade of pathology. CAAs were observed in 2/38 children (5.3%) in the IVIG group and 1/37 children (2.7%) in the methylprednisolone group at 6-month follow- up (difference proportion 0.75; 95% confidence interval (CI) -0.05 to 1.0; p = 0.39). Interpretation Methylprednisolone alone may be an acceptable first-line treatment as left ventricular systolic dysfunction and clinical/laboratory evidence for inflammation quickly resolved in all children. However, our findings need further confirmation through larger studies as our sample size is likely to be of insufficient power to address rare clinically relevant adverse outcomes.

Background The emergence of paediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS) led to the widespread use of anti-inflammatory treatments in the absence of evidence from randomised controlled trials (RCTs). We aimed to assess the effectiveness of intravenous methylprednisolone compared with intravenous immunoglobulins.Methods This is an open-label, multicentre, two-arm RCT done at ten hospitals in Switzerland in children younger than 18 years hospitalised with PIMS-TS (defined as age <18 years; fever and biochemical evidence of inflammation, and single or multiorgan dysfunction; microbiologically proven or putative contact with SARS-CoV-2; and exclusion of any other probable disease). Patients were randomly assigned 1:1 to intravenous methylprednisolone (10 mg/kg per day for 3 days) or intravenous immunoglobulins (2 g/kg as a single dose). The primary outcome was length of hospital stay censored at day 28, death, or discharge. Secondary outcomes included proportion and duration of organ support. Analyses were done by intention-to-treat. The study was registered with Swiss National Clinical Trials Portal (SNCTP000004720) and ClinicalTrials.gov (NCT04826588). Findings Between May 21, 2021, and April 15, 2022, 75 patients with a median age of 9.1 years (IQR 6.2-12.2) were included in the intention-to-treat population (37 in the methylprednisolone group and 38 in the intravenous immunoglobulins group). The median length of hospital stay was 6.0 days (IQR 4.0-8.0) in the methylprednisolone group and 6.0 days (IQR 5.0-8.8) in the intravenous immunoglobulins group (estimated effect size -0.037 of the log10 transformed times, 95% CI -0.13 to 0.065, p=0.42). Fewer patients in the methylprednisolone group (ten [27%] of 37) required respiratory support compared with the intravenous immunoglobulin group (21 [55%] of 38, p=0.025). Need and duration of inotropes, admission to intensive care units, cardiac events after baseline, and major bleeding and thrombotic events were not significantly different between the study groups. Interpretation In this RCT, treatment with methylprednisolone in children with PIMS-TS did not significantly affect the length of hospital stay compared with intravenous immunoglobulins. Intravenous methylprednisolone could be an acceptable first-line treatment in children with PIMS-TS.Funding NOMIS Foundation, Vontobel Foundation, and Gaydoul Foundation.Copyright (c) 2023 Elsevier Ltd. All rights reserved.

IntroductionIn 2020, a new disease entitled Pediatric Inflammatory Multisystem Syndrome temporally associated with COVID-19 (PIMS-TS), or Multisystem Inflammatory Syndrome in Children (MIS-C), emerged, with thousands of children affected globally. There is no available evidence based on randomized controlled trials (RCT) to date on the two most commonly used immunomodulatory treatments, intravenous immunoglobulins (IVIG) and corticosteroids. Therefore, the Swissped RECOVERY trial was conducted to assess whether intravenous (IV) methylprednisolone shortens hospital length of stay compared with IVIG.Methods and AnalysisSwissped RECOVERY is an ongoing investigator-initiated, open-label, multicenter two-arm RCT in children and adolescents <18 years hospitalized with a diagnosis of PIMS-TS. The trial is recruiting at 10 sites across Switzerland. Patients diagnosed with PIMS-TS are randomized 1:1 to methylprednisolone IV (10 mg/kg/day for 3 days) or IVIG (2 g/kg as a single dose). The primary outcome is hospital length of stay censored at day 28, death, or discharge (whichever is first). The target total sample size is ~80 patients 1:1 randomized to each study arm. Ancillary and exploratory studies on inflammation, vaccination acceptance and coverage, long-term outcomes, and healthcare costs are pre-planned.SignificanceCurrently, robust trial evidence for the treatment of PIMS-TS is lacking, with a controversy surrounding the use of corticosteroids vs. IVIG. This trial will provide evidence for the effectiveness and safety of these two treatments.Ethics and DisseminationThe study protocol, which was designed based on the U.K. RECOVERY trial, the patient information and consent forms, and other study-specific study documents were approved by the local ethics committees (Project ID: 2021-00362).Registration DetailsThe study is registered on the Swiss National Clinical Trials Portal (SNCTP000004720) and Clinicaltrials.gov (NCT 04826588).

The impact of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic on pediatric intensive care units (PICUs) is difficult to quantify. We conducted an observational study in all eight Swiss PICUs between 02/24/2020 and 06/15/2020 to characterize the logistical and medical aspects of the pandemic and their impact on the management of the Swiss PICUs. The nine patients admitted to Swiss PICUs during the study period suffering from pediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS) and constituting 14% (9/63) of all SARS-CoV-2 positive hospitalized patients in Swiss children's hospitals caused a higher workload [total Nine Equivalents of nursing Manpower use Score (NEMS) points, p = 0.0008] and were classified to higher workload categories (p < 0.0001) than regular PICU patients (n = 4,881) admitted in 2019. The comparison of the characteristics of the eight Swiss PICUs shows that they were confronted by different organizational issues arising from temporary regulations put in place by the federal council. These general regulations had different consequences for the eight individual PICUs due to the differences between the PICUs. In addition, the temporal relationship of these different regulations influenced the available PICU resources, dependent on the characteristics of the individual PICUs. As pandemic continues, reflecting and learning from experience is essential to reduce workload, optimize bed occupancy and manage resources in each individual PICU. In a small country as Switzerland, with a relatively decentralized health care local differences between PICUs are considerable and should be taken into account when making policy decisions.

The impact of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic on pediatric intensive care units (PICUs) is difficult to quantify. We conducted a retrospective cohort study in all eight Swiss PICUs between 02-24-2020 and 06-15-2020 to characterize the logistical and medical aspects of the pandemic and their impact on Swiss PICUs. Out of the 1113 patients under the age of 20, who were tested positive for SARS-CoV-2 in Switzerland during the study period, 6% (63/1113) had to be hospitalized. All nine patients requiring intensive care had pediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS) and constituted 14% (9/63) of all hospitalized patients. The patients presented with multiple organ dysfunction, needed longer stays in PICUs ( p < 0.0001) and longer positive pressure ventilation ( p = 0.025) than the regular PICU patients. They caused a higher workload (total Nine Equivalents of nursing Manpower use Score (NEMS) points, p = 0.0008) and were classified to higher workload categories (p < 0.0001) than regular PICU patients (n = 4881) in 2019. PICU burden was also influenced by different logistical factors such as cancellation of planned surgeries and staff recruitment to adult wards. Conclusions The different logistical and medical factors associated with the pandemic influenced the workload of the individual PICUs differently, depending on the presence of the single factors and their temporal relationship. The experiences of the study period help further to organize Swiss PICUs during the current pandemic to reduce the workload and optimize occupancy.

Background: Following the spread of the coronavirus disease 2019 (COVID-19) pandemic a new disease entity emerged, defined as Pediatric Inflammatory Multisystem Syndrome temporally associated with COVID-19 (PIMS-TS), or Multisystem Inflammatory Syndrome in Children (MIS-C). In the absence of trials, evidence for treatment remains scarce. Purpose: To develop best practice recommendations for the diagnosis and treatment of children with PIMS-TS in Switzerland. It is acknowledged that the field is changing rapidly, and regular revisions in the coming months are pre-planned as evidence is increasing. Methods: Consensus guidelines for best practice were established by a multidisciplinary group of Swiss pediatric clinicians with expertise in intensive care, immunology/rheumatology, infectious diseases, hematology, and cardiology. Subsequent to literature review, four working groups established draft recommendations which were subsequently adapted in a modified Delphi process. Recommendations had to reach >80% agreement for acceptance. Results: The group achieved agreement on 26 recommendations, which specify diagnostic approaches and interventions across anti-inflammatory, anti-infectious, and support therapies, and follow-up for children with suspected PIMS-TS. A management algorithm was derived to guide treatment depending on the phenotype of presentation, categorized into PIMS-TS with (a) shock, (b) Kawasaki-disease like, and (c) undifferentiated inflammatory presentation. Conclusion: Available literature on PIMS-TS is limited to retrospective or prospective observational studies. Informed by these cohort studies and indirect evidence from other inflammatory conditions in children and adults, as well as guidelines from international health authorities, the Swiss PIMS-TS recommendations represent best practice guidelines based on currently available knowledge to standardize treatment of children with suspected PIMS-TS. Given the absence of high-grade evidence, regular updates of the recommendations will be warranted, and participation of patients in trials should be encouraged.

Most children with a SARS-CoV-2 infection are asymptomatic or exhibit mild symptoms. However, a small number of children develop features of substantial inflammation temporarily related to the COVID-19 also called multisystem inflammatory syndrome in children (MIS-C) or pediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS), clinically similar to Kawasaki disease, toxic shock syndrome and hemophagocytic lymphohistiocytosis (HLH). It is well-known that genetic pre-disposition plays an important role in virally-triggered diseases such as Epstein-Barr virus (EBV)-associated HLH, while this has not yet been established for patients with MIS-C. Here we describe a male patient fulfilling the diagnostic criteria of MIS-C, who was initially treated according to current consensus guidelines. Presence of hypofibrinogenemia, normal lymphocyte counts and C-reactive protein, but substantial hyperferritinemia distinguish this patient from others with MIS-C. The clinical course following initial presentation with acute respiratory distress syndrome was marked by fatal liver failure in the context of EBV-associated HLH despite treatment with steroids, intravenous immunoglobulins, interleukin (IL)-1 receptor blockade and eventually HLH-directed treatment. X-linked lymphoproliferative disease type 1 (XLP1), a subtype of primary HLH was diagnosed in this patient post-mortem. This case report highlights the importance of including HLH in the differential diagnosis in MIS-C with severe disease course to allow specific, risk-adapted treatment and genetic counseling.

This letter is meant to inform the community of pediatricians/pediatric intensivists that we suspect SARS-CoV-2 to cause life-threatening bronchiolitis in infants and we therefore suggest maintaining a high level of suspicion of COVID-19, irrespective of an initially negative SARS-CoV-2 RT-PCR testing, when other causes of bronchiolitis are unidentifiable in young children. Hosted file200626 Letter Andre et al.doc available at https://authorea.com/users/341042/articles/468017can-sars

Natural killer (NK) cells belong to innate lymphoid immune cells that contribute to antitumor responses without requiring prior sensitization. There is strong evidence that NK cells may induce graft-vs-leukemia (GvL) effect without causing graft-vs-host disease (GvHD), and significant efforts are currently undertaken to design and optimize NK cell-based immunotherapeutic strategies against human cancers, particularly against leukemias. However, the results of a number of adoptive NK cell transfer studies have been somewhat disappointing. This may be explained with difficulties in integrating the sequentially appearing activating signals into a directed NK cell response and may also be due to the lack of longevity of the transferred NK cells. The recent understanding that NK cells share developmental and homeostatic properties with T cells led to the hypothesis that NK cells should be able to elicit an (antigen-specific) recall response and should as such theoretically confer the potential for self-renewal and clonal expansion which may translate into longevity. Although immunological memory is per se a hallmark of the adaptive immune system, recent studies performed in mice and humans indicate that NK cells may indeed acquire (under certain conditions) features of adaptive immune cells. This review pursues the aim of providing a general understanding of the cellular and molecular aspects of various forms of memory NK cells with inherent antitumor properties and attempt to describe the translation of this knowledge into recent clinical adoptive transfer protocols with the aim of optimally harnessing the memory NK cell response to certain leukemias.

Fascinating earlier evidence suggests an intrinsic capacity of human natural killer (NK) cells to acquire adaptive immune features in the context of cytomegalovirus (CMV) infection or pro-inflammatory cytokine stimulation. Since the role of memory NK cells in cancer has so far remained elusive and adoptive NK cell transfer in relapsing pediatric acute B cell precursor leukemia (BCP-ALL) patients awaits improvement, we asked the question whether tumor-priming could promote the generation of memory NK cells with enhanced graft-vs.-leukemia (GvL) reactivity. Here, we provide substantial evidence that priming of naive human NK cells with pediatric acute B cell leukemia or acute myeloid leukemia specimens induces a functional conversion to tumor-induced memory-like (TIML)-NK cells displaying a heightened tumor-specific cytotoxicity and enhanced perforin synthesis. Cell cycles analyses reveal that tumor-priming sustainably alters the balance between NK cell activation and apoptosis in favor of survival. In addition, gene expression patterns differ between TIML- and cytokine-induced memory-like (CIML)-NK cells with the magnitude of regulated genes being distinctly higher in TIML-NK cells. As such, the tumor-induced conversion of NK cells triggers the emergence of a so far unacknowledged NK cell differentiation stage that might promote GvL effects in the context of adoptive cell transfer.

The treatment of leukemia/lymphoma by chimeric antigen receptor (CAR) redirected T cells with specificity for CD19 induced complete remissions in the majority of patients, with a realistic hope for cure. However, recent follow-up data revealed a substantial risk of relapse through leukemic cells that lack the CAR targeted antigen. In this situation, a bispecific CAR with binding domains for CD19 and CD20 is aimed at recognizing leukemic cells with only one cognate antigen. The anti-CD20-CD19 bispecific CAR induced a full T-cell response upon engagement of CD19 or CD20 on target cells showing a true "OR" gate recognition in redirecting T-cell activation. T cells with the anti-CD20-CD19 CAR efficiently killed patients' chronic lymphocytic leukemia cells in vitro. The bispecific CAR T cells cleared pediatric acute lymphocytic leukemia with a mixed CD19+CD20+/CD20- phenotype from the blood and bone marrow of transplanted mice, while anti-CD20 CAR T cells left CD20- leukemic cells behind without curing the disease. Data indicate the superior anti-leukemic activity in the control of leukemia, implying that the anti-CD20-CD19 bispecific CAR T cells may reduce the risk of relapse through antigen-loss leukemic cells in the long term.

NK cells are functionally controlled by the killer immunoglobulin-like receptor (KIR) family that comprises inhibitory (iKIR) and activating (aKIR) members. Genetic association studies suggest that donors expressing aKIRs next to iKIRs will be superior donors in the setting of hematopoietic stem cell transplantation of patients with leukemia. However, contrary evidence states that aKIR expression may be irrelevant or even detrimental. Using a complex methodology incorporating KIR-Q-PCR, double fluorescence and viSNE analysis, we characterized subset distribution patterns and functionality in haplotype A donors which lack aKIRs and haplotype B donors that express a variety of B-specific genes. Here, we show that the alloreactive KIR2DS1(+) NK cell subset in HLA-C1/C2 donors is highly responsive towards C2-expressing targets but quantitatively small and as such does not significantly contribute to cytotoxicity. Thus, we fail to find a direct link between haplotype allocation status and NK cell cytotoxicity at least in HLA-C1/C2 heterozygous donors.

Natural killer (NK) cells play an important role in surveillance and elimination of malignant cells. Their spontaneous cytotoxicity was first demonstrated in vitro against leukemia cell lines, and NK cells might play a crucial role in the therapy of leukemia. NK cell activity is controlled by an array of germ line-encoded activating and inhibitory receptors, as well as modulating coreceptors. This biologic feature can be exploited in allogeneic cell therapy, and the recognition of "missing-self" on target cells is crucial for promoting NK cell-mediated graft-versus-leukemia effects. In this regard, NK cells that express an inhibitory killer immunoglobulin-like receptor (iKIR) for which the respective major histocompatibility complex class I ligand is absent on leukemic target cells can exert alloreactivity in vitro and in vivo. Several models regarding potential donor-patient constellations have been described that have demonstrated the clinical benefit of such alloreactivity of the donor-derived NK cell system in patients with adult acute myeloid leukemia and pediatric B-cell precursor acute lymphoblastic leukemia after allogeneic stem cell transplantation. Moreover, adoptive transfer of mature allogeneic NK cells in the nontransplant or transplant setting has been shown to be safe and feasible, whereas its effectivity needs further evaluation. NK cell therapy can be further improved by optimal donor selection based on phenotypic and genotypic properties, by adoptive transfer of NK cells with ex vivo or in vivo cytokine stimulation, by the use of antibodies to induce antibody-dependent cellular cytotoxicity or to block iKIRs, or by transduction of chimeric antigen receptors.