Engineering of CD34+ progenitor-derived natural killer cells with higher-affinity CD16a for enhanced antibody-dependent cellular cytotoxicity.

BACKGROUND AIMS: Natural killer (NK) cell transfer is a promising cellular immunotherapy for cancer. Previously, we developed a robust method to generate large NK cell numbers from CD34+ hematopoietic stem and progenitor cells (HSPCs), which exhibit strong anti-tumor activity. However, since these cells express low levels of the Fc receptor CD16a in vitro, antibody-dependent cellular cytotoxicity (ADCC) by these cells is limited. To broaden clinical applicability of our HSPC-NK cells toward less NK-sensitive malignancies, we aimed to improve ADCC through CD16a transduction. METHODS: Using wildtype and S197P mutant greater-affinity (both with V158) CD16a retroviral transgenes (i.e., a cleavable and noncleavable CD16a upon stimulation), we generated CD16a HSPC-transduced NK cells, with CD34+ cells isolated from umbilical cord blood (UCB) or peripheral blood after G-CSF stem cell mobilization (MPB). CD16a expressing NK cells were enriched using flow cytometry-based cell sorting. Subsequently, phenotypic analyses and functional assays were performed to investigate natural cytotoxicity and ADCC activity. RESULTS: Mean transduction efficiency was 34% for UCB-derived HSPCs and 20% for MPB-derived HSPCs, which was enriched by flow cytometry-based cell sorting to >90% for both conditions. Expression of the transgene remained stable during the entire NK expansion cell generation process. Proliferation and differentiation of HSPCs were not hampered by the transduction process, resulting in effectively differentiated CD56+ NK cells after 5 weeks. Activation of the HSPC-derived NK cells resulted in significant shedding of wildtype CD16a transcribed from the endogenous gene, but not of the noncleavable mutant CD16a protein expressed from the transduced construct. The mean increase of CD107+IFNγ+ expressing NK cells after inducing ADCC was tenfold in enriched noncleavable CD16a HSPC-NK cells. Killing capacity of CD16a-transduced NK cells was significantly improved after addition of a tumor-targeting antibody in tumor cell lines and primary B-cell leukemia and lymphoma cells compared to unmodified HSPC-NK cells. CONCLUSIONS: Together, these data demonstrate that the applicability of adoptive NK cell immunotherapy may be broadened to less NK-sensitive malignancies by upregulation of CD16a expression in combination with the use of tumor-targeting monoclonal antibodies.

Human CD34+-derived complete plasmacytoid and conventional dendritic cell vaccine effectively induces antigen-specific CD8+ T cell and NK cell responses in vitro and in vivo.

Allogeneic stem cell transplantation (alloSCT) can be curative for hemato-oncology patients due to effective graft-versus-tumor immunity. However, relapse remains the major cause of treatment failure, emphasizing the need for adjuvant immunotherapies. In this regard, post-transplantation dendritic cell (DC) vaccination is a highly interesting strategy to boost graft-versus-tumor responses. Previously, we developed a clinically applicable protocol for simultaneous large-scale generation of end-stage blood DC subsets from donor-derived CD34+ stem cells, including conventional type 1 and 2 DCs (cDC1s and cDC2s), and plasmacytoid DCs (pDCs). In addition, the total cultured end-product (DC-complete vaccine), also contains non-end-stage-DCs (i.e. non-DCs). In this study, we aimed to dissect the phenotypic identity of these non-DCs and their potential immune modulatory functions on the potency of cDCs and pDCs in stimulating tumor-reactive CD8+ T and NK cell responses, in order to obtain rationale for clinical translation of our DC-complete vaccine. The non-DC compartment was heterogeneous and comprised of myeloid progenitors and (immature) granulocyte- and monocyte-like cells. Importantly, non-DCs potentiated toll-like receptor-induced DC maturation, as reflected by increased expression of co-stimulatory molecules and enhanced cDC-derived IL-12 and pDC-derived IFN-α production. Additionally, antigen-specific CD8+ T cells effectively expanded upon DC-complete vaccination in vitro and in vivo. This effect was strongly augmented by non-DCs in an antigen-independent manner. Moreover, non-DCs did not impair in vitro DC-mediated NK cell activation, degranulation nor cytotoxicity. Notably, in vivo i.p. DC-complete vaccination activated i.v. injected NK cells. Together, these data demonstrate that the non-DC compartment potentiates DC-mediated activation and expansion of antigen-specific CD8+ T cells and do not impair NK cell responses in vitro and in vivo. This underscores the rationale for further clinical translation of our CD34+-derived DC-complete vaccine in hemato-oncology patients post alloSCT.

Analytical assay validation for acute myeloid leukemia measurable residual disease assessment by multiparametric flow cytometry.

BACKGROUND: Measurable residual disease (MRD) assessed by multiparametric flow cytometry (MFC) has gained importance in clinical decision-making for acute myeloid leukemia (AML) patients. However, complying with the recent In Vitro Diagnostic Regulations (IVDR) in Europe and Food and Drug Administration (FDA) guidance in the United States requires rigorous validation prior to their use in investigational clinical trials and diagnostics. Validating AML MRD-MFC assays poses challenges due to the unique underlying disease biology and paucity of patient specimens. In this study, we describe an experimental framework for validation that meets regulatory expectations. METHODS: Our validation efforts focused on evaluating assay accuracy, analytical specificity, analytical and functional sensitivity (limit of blank (LoB), detection (LLoD) and quantitation (LLoQ)), precision, linearity, sample/reagent stability and establishing the assay background frequencies. RESULTS: Correlation between different MFC methods was highly significant (r = 0.99 for %blasts and r = 0.93 for %LAIPs). The analysis of LAIP specificity accurately discriminated from negative control cells. The assay demonstrated a LoB of 0.03, LLoD of 0.04, and LLoQ of 0.1%. Precision experiments yielded highly reproducible results (Coefficient of Variation <20%). Stability experiments demonstrated reliable measurement of samples up to 96 h from collection. Furthermore, the reference range of LAIP frequencies in non-AML patients was below 0.1%, ranging from 0.0% to 0.04%. CONCLUSION: In this manuscript, we present the validation of an AML MFC-MRD assay using BM/PB patient specimens, adhering to best practices. Our approach is expected to assist other laboratories in expediting their validation activities to fulfill recent health authority guidelines.

Saponin-based adjuvants enhance antigen cross-presentation in human CD11c+ CD1c+ CD5- CD163+ conventional type 2 dendritic cells.

BACKGROUND: Adjuvants are key for effective vaccination against cancer and chronic infectious diseases. Saponin-based adjuvants (SBAs) are unique among adjuvants in their ability to induce robust cell-mediated immune responses in addition to antibody responses. Recent preclinical studies revealed that SBAs induced cross-presentation and lipid bodies in otherwise poorly cross-presenting CD11b+ murine dendritic cells (DCs). METHOD: Here, we investigated the response of human DC subsets to SBAs with RNA sequencing and pathway analyses, lipid body induction visualized by laser scanning microscopy, antigen translocation to the cytosol, and antigen cross-presentation to CD8+ T cells. RESULTS: RNA sequencing of SBA-treated conventional type 1 DC (cDC1) and type 2 DC (cDC2) subsets uncovered that SBAs upregulated lipid-related pathways in CD11c+ CD1c+ cDC2s, especially in the CD5- CD163+ CD14+ cDC2 subset. Moreover, SBAs induced lipid bodies and enhanced endosomal antigen translocation into the cytosol in this particular cDC2 subset. Finally, SBAs enhanced cross-presentation only in cDC2s, which requires the CD163+ CD14+ cDC2 subset. CONCLUSIONS: These data thus identify the CD163+ CD14+ cDC2 subset as the main SBA-responsive DC subset in humans and imply new strategies to optimize the application of saponin-based adjuvants in a potent cancer vaccine.

Flow cytometric analysis of myelodysplasia: Pre-analytical and technical issues-Recommendations from the European LeukemiaNet.

BACKGROUND: Flow cytometry (FCM) aids the diagnosis and prognostic stratification of patients with suspected or confirmed myelodysplastic syndrome (MDS). Over the past few years, significant progress has been made in the FCM field concerning technical issues (including software and hardware) and pre-analytical procedures. METHODS: Recommendations are made based on the data and expert discussions generated from 13 yearly meetings of the European LeukemiaNet international MDS Flow working group. RESULTS: We report here on the experiences and recommendations concerning (1) the optimal methods of sample processing and handling, (2) antibody panels and fluorochromes, and (3) current hardware technologies. CONCLUSIONS: These recommendations will support and facilitate the appropriate application of FCM assays in the diagnostic workup of MDS patients. Further standardization and harmonization will be required to integrate FCM in MDS diagnostic evaluations in daily practice.

Clinical application of flow cytometry in patients with unexplained cytopenia and suspected myelodysplastic syndrome: A report of the European LeukemiaNet International MDS-Flow Cytometry Working Group.

This article discusses the rationale for inclusion of flow cytometry (FCM) in the diagnostic investigation and evaluation of cytopenias of uncertain origin and suspected myelodysplastic syndromes (MDS) by the European LeukemiaNet international MDS Flow Working Group (ELN iMDS Flow WG). The WHO 2016 classification recognizes that FCM contributes to the diagnosis of MDS and may be useful for prognostication, prediction, and evaluation of response to therapy and follow-up of MDS patients.

Multiparameter flow cytometry in the evaluation of myelodysplasia: Analytical issues: Recommendations from the European LeukemiaNet/International Myelodysplastic Syndrome Flow Cytometry Working Group.

Multiparameter flow cytometry (MFC) is one of the essential ancillary methods in bone marrow (BM) investigation of patients with cytopenia and suspected myelodysplastic syndrome (MDS). MFC can also be applied in the follow-up of MDS patients undergoing treatment. This document summarizes recommendations from the International/European Leukemia Net Working Group for Flow Cytometry in Myelodysplastic Syndromes (ELN iMDS Flow) on the analytical issues in MFC for the diagnostic work-up of MDS. Recommendations for the analysis of several BM cell subsets such as myeloid precursors, maturing granulocytic and monocytic components and erythropoiesis are given. A core set of 17 markers identified as independently related to a cytomorphologic diagnosis of myelodysplasia is suggested as mandatory for MFC evaluation of BM in a patient with cytopenia. A myeloid precursor cell (CD34+ CD19- ) count >3% should be considered immunophenotypically indicative of myelodysplasia. However, MFC results should always be evaluated as part of an integrated hematopathology work-up. Looking forward, several machine-learning-based analytical tools of interest should be applied in parallel to conventional analytical methods to investigate their usefulness in integrated diagnostics, risk stratification, and potentially even in the evaluation of response to therapy, based on MFC data. In addition, compiling large uniform datasets is desirable, as most of the machine-learning-based methods tend to perform better with larger numbers of investigated samples, especially in such a heterogeneous disease as MDS.

Reprogramming of myeloid cells and their progenitors in patients with non-medullary thyroid carcinoma.

Myeloid cells, crucial players in antitumoral defense, are affected by tumor-derived factors and treatment. The role of myeloid cells and their progenitors prior to tumor infiltration is poorly understood. Here we show single-cell transcriptomics and functional analyses of the myeloid cell lineage in patients with non-medullary thyroid carcinoma (TC) and multinodular goiter, before and after treatment with radioactive iodine compared to healthy controls. Integrative data analysis indicates that monocytes of TC patients have transcriptional upregulation of antigen presentation, reduced cytokine production capacity, and overproduction of reactive oxygen species. Interestingly, these cancer-related pathological changes are partially removed upon treatment. In bone marrow, TC patients tend to shift from myelopoiesis towards lymphopoiesis, reflected in transcriptional differences. Taken together, distinct transcriptional and functional changes in myeloid cells arise before their infiltration of the tumor and are already initiated in bone marrow, which suggests an active role in forming the tumor immune microenvironment.

Anti-Tumor Potency of Short-Term Interleukin-15 Dendritic Cells Is Potentiated by In Situ Silencing of Programmed-Death Ligands.

Dendritic cell (DC) vaccines have proven to be a valuable tool in cancer immune therapy. With several DC vaccines being currently tested in clinical trials, knowledge about their therapeutic value has been significantly increased in the past decade. Despite their established safety, it has become clear that objective clinical responses are not yet robust enough, requiring further optimization. Improvements of this advanced therapy medicinal product encompass, among others, regulating their immune stimulating capacity by in situ gene engineering, in addition to their implementation in combination therapy regimens. Previously, we have reported on a superior monocyte-derived DC preparation, including interleukin-15, pro-inflammatory cytokines and immunological danger signals in the culture process. These so-called IL-15 DCs have already proven to exhibit several favorable properties as cancer vaccine. Evolving research into mechanisms that could further modulate the immune response towards cancer, points to programmed death-1 as an important player that dampens anti-tumor immunity. Aiming at leveraging the immunogenicity of DC vaccines, we hypothesized that additional implementation of the inhibitory immune checkpoint molecules programmed death-ligand (PD-L)1 and PD-L2 in IL-15 DC vaccines would exhibit superior stimulatory potential. In this paper, we successfully implemented PD-L silencing at the monocyte stage in the 3-day IL-15 DC culture protocol resulting in substantial downregulation of both PD-L1 and PD-L2 to levels below 30%. Additionally, we validated that these DCs retain their specific characteristics, both at the level of phenotype and interferon gamma secretion. Evaluating their functional characteristics, we demonstrate that PD-L silencing does not affect the capacity to induce allogeneic proliferation. Ultimately designed to induce a durable tumor antigen-specific immune response, PD-L silenced IL-15 DCs were capable of surpassing PD-1-mediated inhibition by antigen-specific T cells. Further corroborating the superior potency of short-term IL-15 DCs, the combination of immune stimulatory components during DC differentiation and maturation with in situ checkpoint inhibition supports further clinical translation.

OMIP - 081: A new 21-monoclonal antibody 10-color panel for diagnostic polychromatic immunophenotyping.

The 10-color panel consisting of 21 monoclonal antibodies (mAbs) is developed as a one-tube panel to detect leukemia and lymphoma cells in all hematopoietic cell lineages. In particular, this tube is mentioned for a fast screening to identify aberrant cells in samples suspected for malignant cell localization and to enable comprehensive immunophenotyping of samples with low cell counts. The panel contains mAbs for selection of the populations and mAbs against target antigens on the various hematopoietic maturation stages. Due to the limited number of PMTs in most used flow cytometers for clinical purposes, stacking of conjugates in one color is needed to include all relevant markers for simultaneous analysis of the aberrant cells. The 21-mAb panel is tested on peripheral blood (PB), and bone marrow (BM) samples and enables an efficient and correct identification of hematological malignancies. This panel improves the diagnostic potential.

CD34+ progenitor-derived NK cell and gemcitabine combination therapy increases killing of ovarian cancer cells in NOD/SCID/IL2Rgnull mice.

Combining natural killer (NK) cell adoptive transfer with tumor-sensitizing chemotherapy is an attractive approach against recurrent ovarian cancer (OC), as OC is sensitive to NK cell-mediated immunity. Previously, we showed that CD34+ hematopoietic progenitor cell (HPC)-derived NK cells can kill OC cells in vitro and inhibit OC tumor growth in mice. Here, we investigated the potential of HPC-NK cell therapy combined with chemotherapeutic gemcitabine (used in recurrent OC patients) against OC. We examined the phenotypical, functional, and cytotoxic effects of gemcitabine on HPC-NK cells and/or OC cells in vitro and in OC-bearing mice. To this end, we treated OC cells and/or HPC-NK cells with or without gemcitabine and analyzed the phenotype, cytokine production, and anti-tumor reactivity. We found that gemcitabine did not affect the phenotype and functionality of HPC-NK cells, while on OC cells expression of NK cell activating ligands and death receptors was upregulated. Although gemcitabine pre-treatment of OC cells did not improve the functionality of HPC-NK cells, importantly, HPC-NK cells and gemcitabine additively killed OC cells in vitro. Similarly, combined HPC-NK cell and gemcitabine treatment additively decreased tumor growth in OC-bearing mice. Collectively, our results indicate that combination therapy of HPC-NK cells and gemcitabine results in augmented OC killing in vitro and in vivo. This provides a rationale for exploring this therapeutic strategy in patients with recurrent OC.

Clinically applicable CD34+-derived blood dendritic cell subsets exhibit key subset-specific features and potently boost anti-tumor T and NK cell responses.

Allogeneic stem cell transplantation (alloSCT), following induction chemotherapy, can be curative for hemato-oncology patients due to powerful graft-versus-tumor immunity. However, disease recurrence remains the major cause of treatment failure, emphasizing the need for potent adjuvant immunotherapy. In this regard, dendritic cell (DC) vaccination is highly attractive, as DCs are the key orchestrators of innate and adaptive immunity. Natural DC subsets are postulated to be more powerful compared with monocyte-derived DCs, due to their unique functional properties and cross-talk capacity. Yet, obtaining sufficient numbers of natural DCs, particularly type 1 conventional DCs (cDC1s), is challenging due to low frequencies in human blood. We developed a clinically applicable culture protocol using donor-derived G-CSF mobilized CD34+ hematopoietic progenitor cells (HPCs) for simultaneous generation of high numbers of cDC1s, cDC2s and plasmacytoid DCs (pDCs). Transcriptomic analyses demonstrated that these ex vivo-generated DCs highly resemble their in vivo blood counterparts. In more detail, we demonstrated that the CD141+CLEG9A+ cDC1 subset exhibited key features of in vivo cDC1s, reflected by high expression of co-stimulatory molecules and release of IL-12p70 and TNF-α. Furthermore, cDC1s efficiently primed alloreactive T cells, potently cross-presented long-peptides and boosted expansion of minor histocompatibility antigen-experienced T cells. Moreover, they strongly enhanced NK cell activation, degranulation and anti-leukemic reactivity. Together, we developed a robust culture protocol to generate highly functional blood DC subsets for in vivo application as tailored adjuvant immunotherapy to boost innate and adaptive anti-tumor immunity in alloSCT patients.

A trispecific killer engager molecule against CLEC12A effectively induces NK-cell mediated killing of AML cells.

The low 5-year survival rate for patients with acute myeloid leukemia (AML), primarily caused due to disease relapse, emphasizes the need for better therapeutic strategies. Disease relapse is facilitated by leukemic stem cells (LSCs) that are resistant to standard chemotherapy and promote tumor growth. To target AML blasts and LSCs using natural killer (NK) cells, we have developed a trispecific killer engager (TriKETM) molecule containing a humanized anti-CD16 heavy chain camelid single-domain antibody (sdAb) that activates NK cells, an IL-15 molecule that drives NK-cell priming, expansion and survival, and a single-chain variable fragment (scFv) against human CLEC12A (CLEC12A TriKE). CLEC12A is a myeloid lineage antigen that is highly expressed by AML cells and LSCs, but not expressed by normal hematopoietic stem cells (HSCs), thus minimizing off-target toxicity. The CLEC12A TriKE induced robust NK-cell specific proliferation, enhanced NK-cell activation, and killing of both AML cell lines and primary patient-derived AML blasts in vitro while sparing healthy HSCs. Additionally, the CLEC12A TriKE was able to reduce tumor burden in preclinical mouse models. These findings highlight the clinical potential of the CLEC12A TriKE for the effective treatment of AML.

TIGIT blockade enhances functionality of peritoneal NK cells with altered expression of DNAM-1/TIGIT/CD96 checkpoint molecules in ovarian cancer.

Advanced ovarian cancer (OC) patients have a poor 5-year survival of only 28%, emphasizing the medical need for improved therapies. Adjuvant immunotherapy could be an attractive approach since OC is an immunogenic disease and the presence of tumor-infiltrating lymphocytes has shown to positively correlate with patient survival. Among these infiltrating lymphocytes are natural killer (NK) cells, key players involved in tumor targeting, initiated by signaling via activating and inhibitory receptors. Here, we investigated the role of the DNAM-1/TIGIT/CD96 axis in the anti-tumor response of NK cells toward OC. Ascites-derived NK cells from advanced OC patients showed lower expression of activating receptor DNAM-1 compared to healthy donor peripheral blood NK cells, while inhibitory receptor TIGIT and CD96 expression was equal or higher, respectively. This shift to a more inhibitory phenotype could also be induced in vitro by co-culturing healthy donor NK cells with OC tumor spheroids, and in vivo on intraperitoneally infused NK cells in SKOV-3 OC bearing NOD/SCID-IL2Rγnull (NSG) mice. Interestingly, TIGIT blockade enhanced degranulation and interferon gamma (IFNγ) production of healthy donor CD56dim NK cells in response to OC tumor cells, especially when DNAM-1/CD155 interactions were in place. Importantly, TIGIT blockade boosted functional responsiveness of CD56dim NK cells of OC patients with a baseline reactivity against SKOV-3 cells. Overall, our data show for the first time that checkpoint molecules TIGIT/DNAM-1/CD96 play an important role in NK cell responsiveness against OC, and provides rationale for incorporating TIGIT interference in NK cell-based immunotherapy in OC patients.

PD-L1 siRNA-mediated silencing in acute myeloid leukemia enhances anti-leukemic T cell reactivity.

Acute myeloid leukemia (AML) is an immune-susceptible malignancy, as demonstrated by its responsiveness to allogeneic stem cell transplantation (alloSCT). However, by employing inhibitory signaling pathways, including PD-1/PD-L1, leukemia cells suppress T cell-mediated immune attack. Notably, impressive clinical efficacy has been obtained with PD-1/PD-L1 blocking antibodies in cancer patients. Yet, these systemic treatments are often accompanied by severe toxicity, especially after alloSCT. Here, we investigated RNA interference technology as an alternative strategy to locally interfere with PD-1/PD-L1 signaling in AML. We demonstrated efficient siRNA-mediated PD-L1 silencing in HL-60 and patients' AML cells. Importantly, WT1-antigen T cell receptor+ PD-1+ 2D3 cells showed increased activation toward PD-L1 silenced WT1+ AML. Moreover, PD-L1 silenced AML cells significantly enhanced the activation, degranulation, and IFN-γ production of minor histocompatibility antigen-specific CD8+ T cells. Notably, PD-L1 silencing was equally effective as PD-1 antibody blockade. Together, our study demonstrates that PD-L1 silencing may be an effective strategy to augment AML immune-susceptibility. This provides rationale for further development of targeted approaches to locally interfere with immune escape mechanisms in AML, thereby minimizing severe toxicity. In combination with alloSCT and/or adoptive T cell transfer, this strategy could be very appealing to boost graft-versus-leukemia immunity and improve outcome in AML patients.

Cell composition and expansion strategy can reduce the beneficial effect of AKT-inhibition on functionality of CD8+ T cells.

AKT-inhibition is a promising approach to improve T cell therapies; however, its effect on CD4+ T cells is insufficiently explored. Previously, we and others showed that AKT-inhibition during ex vivo CD8+ T cell expansion facilitates the generation of polyfunctional T cells with stem cell memory-like traits. However, most therapeutic T cell products are generated from lymphocytes, containing CD4+ T cells that can affect CD8+ T cells dependent on the Th-subset. Here, we investigated the effect of AKT-inhibition on CD4+ T cells, during separate as well as total T cell expansions. Interestingly, ex vivo AKT-inhibition preserved the early memory phenotype of CD4+ T cells based on higher CD62L, CXCR4 and CCR7 expression. However, in the presence of AKT-inhibition, Th-differentiation was skewed toward more Th2-associated at the expense of Th1-associated cells. Importantly, the favorable effect of AKT-inhibition on the functionality of CD8+ T cells drastically diminished in the presence of CD4+ T cells. Moreover, also the expansion method influenced the effect of AKT-inhibition on CD8+ T cells. These findings indicate that the effect of AKT-inhibition on CD8+ T cells is dependent on cell composition and expansion strategy, where presence of CD4+ T cells as well as polyclonal stimulation impede the favorable effect of AKT-inhibition.

Intraperitoneal infusion of ex vivo-cultured allogeneic NK cells in recurrent ovarian carcinoma patients (a phase I study).

INTRODUCTION: Recurrent ovarian carcinoma has dismal prognosis, but control of disease and prolonged survival are possible in some patients. The estimated 5-year survival is 46% for all stages of ovarian cancer, and only 28% for metastasized disease. Notably, the majority of women with ovarian cancer are diagnosed with stage III or IV disease with a high recurrence rate. As most women with relapsed or metastatic cancer will die of progressive disease, there is an urgent need for novel therapeutic strategies. The primary aim of our study is to evaluate safety and toxicity of intraperitoneal infusion of ex vivo-expanded natural killer cells (NK), generated from CD34+ umbilical cord blood (UCB) progenitor cells, with and without a preceding non-myeloablative immunosuppressive conditioning regimen in patients suffering from recurrent ovarian cancer. The secondary objectives are to compare the in vivo lifespan, expansion, and biological activity of intraperitoneally infused NK cell products with or without preparative chemotherapy, as well as evaluate effects on disease load. METHODS: In this phase I safety trial, 12 patients who are suffering from recurrent ovarian cancer, detected by a significant rise in serum level of CA-125 on two successive time points, will be included. Prior to UCB-NK cell infusion, a laparoscopy is performed to place a catheter in the peritoneal cavity. The first cohort of three patients will receive a single intraperitoneal infusion of 1.5-3×10 UCB-NK cells, generated ex vivo from CD34+ hematopoietic progenitor cells obtained from an allogeneic UCB unit, without a preparative chemotherapy regimen. The second group of three patients will be treated with a similar dose of UCB-NK cells following a preparative four days non-myeloablative immunosuppressive conditioning regimen with cyclophosphamide and fludarabine (Cy/Flu). If no severe toxicity is seen in these 6 patients, an extension cohort of 6 patients will be included to answer the secondary objectives. DISCUSSION: This study investigates the safety of a promising new cellular therapy in a group of patients with a poor prognosis. Demonstration of safety and in vivo expansion capacity of allogeneic UCB-NK cells in the absence of Cy/Flu pretreatment will provide rationale for UCB-NK cell infusion after regular second-line chemotherapy.

Comprehensive Phenotyping of T Cells Using Flow Cytometry.

The T cell compartment can form a powerful defense against extrinsic (e.g., pathogens) and intrinsic danger (e.g., malignant cells). At the same time, specific subsets of T cells control this process to keep the immune system in check and prevent autoimmunity. A wide variety in T cell functionalities exists, which is dependent on the differentiation and maturation state of the T cells. In this review, we report an overview for the identification of CD4+ T-αß cells (T-helper (Th)1, Th2, Th9, Th17, Th22, and CD4+ regulatory T cells), CD8+ T-αß cells (cytotoxic T lymphocyte (Tc)1, Tc2, Tc9, Tc17, and CD8+ regulatory T cells), and their additional effector memory status (naïve, stem cell memory, central memory, effector memory, and effector) using flow cytometry. These different subsets can be discriminated based on selective extracellular markers, in combination with intracellular transcription factor and/or cytokine stainings. Additionally, identification of very small subsets, including antigen-specific T cells, and important technical considerations of flow cytometry are discussed. Together, this overview can be used for comprehensive phenotyping of a T cell subset of interest. © 2019 International Society for Advancement of Cytometry.

PD-L1 microSPECT/CT Imaging for Longitudinal Monitoring of PD-L1 Expression in Syngeneic and Humanized Mouse Models for Cancer.

Antibodies that block the interaction between programmed death ligand 1 (PD-L1) and PD-1 have shown impressive responses in subgroups of patients with cancer. PD-L1 expression in tumors seems to be a prerequisite for treatment response. However, PD-L1 is heterogeneously expressed within tumor lesions and may change upon disease progression and treatment. Imaging of PD-L1 could aid in patient selection. Previously, we showed the feasibility to image PD-L1+ tumors in immunodeficient mice. However, PD-L1 is also expressed on immune cell subsets. Therefore, the aim of this study was to assess the potential of PD-L1 micro single-photon emission tomography/computed tomography (microSPECT/CT) using radiolabeled PD-L1 antibodies to (i) measure PD-L1 expression in two immunocompetent tumor models (syngeneic mice and humanized mice harboring PD-L1 expressing immune cells) and (ii) monitor therapy-induced changes in tumor PD-L1 expression. We showed that radiolabeled PD-L1 antibodies accumulated preferentially in PD-L1+ tumors, despite considerable uptake in certain normal lymphoid tissues (spleen and lymph nodes) and nonlymphoid tissues (duodenum and brown fat). PD-L1 microSPECT/CT imaging could also distinguish between high and low PD-L1-expressing tumors. The presence of PD-L1+ immune cells did not compromise tumor uptake of the human PD-L1 antibodies in humanized mice, and we demonstrated that radiotherapy-induced upregulation of PD-L1 expression in murine tumors could be monitored with microSPECT/CT imaging. Together, these data demonstrate that PD-L1 microSPECT/CT is a sensitive technique to detect variations in tumor PD-L1 expression, and in the future, this technique may enable patient selection for PD-1/PD-L1-targeted therapy.

Peritoneal NK cells are responsive to IL-15 and percentages are correlated with outcome in advanced ovarian cancer patients.

The demonstration that ovarian carcinoma (OC) is an immunogenic disease, opens opportunities to explore immunotherapeutic interventions to improve clinical outcome. In this regard, NK cell based immunotherapy could be promising as it has been demonstrated that OC cells are susceptible to killing by cytokine-stimulated NK cells. Here, we evaluated whether percentage, phenotype, function and IL-15 responsiveness of ascites-derived natural killer (NK) cells is related to progression-free survival (PFS) and overall survival (OS) of advanced stage OC patients. Generally, a lower percentage of NK cells within the lymphocyte fraction was seen in OC ascites (mean 17.4 ± 2.7%) versus benign peritoneal fluids (48.1 ± 6.8%; p < 0.0001). Importantly, a higher CD56+ NK cell percentage in ascites was associated with a better PFS (p = 0.01) and OS (p = 0.002) in OC patients. Furthermore, the functionality of ascites-derived NK cells in terms of CD107a/IFN-γ activity was comparable to that of healthy donor peripheral blood NK cells, and stimulation with monomeric IL-15 or IL-15 superagonist ALT-803 potently improved their reactivity towards tumor cells. By showing that a higher NK cell percentage is related to better outcome in OC patients and NK cell functionality can be boosted by IL-15 receptor stimulation, a part of NK cell immunity in OC is further deciphered to exploit NK cell based immunotherapy.