Good manufacturing practice production of CD34+ progenitor-derived NK cells for adoptive immunotherapy in acute myeloid leukemia.

Allogeneic natural killer (NK) cell-based immunotherapy is a promising, well-tolerated adjuvant therapeutic approach for acute myeloid leukemia (AML). For reproducible NK cell immunotherapy, a homogenous, pure and scalable NK cell product is preferred. Therefore, we developed a good manufacturing practice (GMP)-compliant, cytokine-based ex vivo manufacturing process for generating NK cells from CD34+ hematopoietic stem and progenitor cells (HSPC). This manufacturing process combines amongst others IL15 and IL12 and the aryl hydrocarbon receptor antagonist StemRegenin-1 (SR1) to generate a consistent and active NK cell product that fits the requirements for NK cell immunotherapy well. The cell culture protocol was first optimized to generate NK cells with required expansion and differentiation capacity in GMP-compliant closed system cell culture bags. In addition, phenotype, antitumor potency, proliferative and metabolic capacity were evaluated to characterize the HSPC-NK product. Subsequently, seven batches were manufactured for qualification of the process. All seven runs demonstrated consistent results for proliferation, differentiation and antitumor potency, and preliminary specifications for the investigational medicinal product for early clinical phase trials were set. This GMP-compliant manufacturing process for HSPC-NK cells (named RNK001 cells) is used to produce NK cell batches applied in the clinical trial 'Infusion of ex vivo-generated allogeneic natural killer cells in combination with subcutaneous IL2 in patients with acute myeloid leukemia' approved by the Dutch Ethics Committee (EudraCT 2019-001929-27).

Effects of antibody concentration on the separation of human natural killer cells in a commercial immunomagnetic separation system.

BACKGROUND: The magnetic separation of a cell population based on cell surface markers is a critical step in many biological and clinical laboratories. In this study, the effect of antibody concentration on the separation of human natural killer cells in a commercial, immunomagnetic cell separation system was investigated. METHODS: Specifically, the degree of saturation of antibody binding sites using a two-step antibody sandwich was quantified. The quantification of the first step, a primary anti-CD56-PE antibody, was achieved through fluorescence intensity measurements using a flow cytometer. The quantification of the second step, an anti-PE-microbeads antibody reagent, was achieved through magnetophoretic mobility measurements using cell tracking velocimetry. RESULTS: From the results of these studies, two different labeling protocols were used to separate CD56+ cells from human, peripheral blood by a Miltenyi Biotech MiniMACS cell separation system. The first of these two labeling protocols was based on company recommendations, whereas the second was based on the results of the saturation studies. The results from these studies demonstrate that the magnetophoretic mobility is a function of both primary and secondary antibody concentrations and that mobility does have an effect on the performance of the separation system. CONCLUSIONS: As the mobility increased due to an increase in bound antibodies, the positive cells were almost completely eliminated from the negative eluent. However, with an increase in bound antibodies, and thus mobility, the total amount of positive cells recovered decreases. It is speculated that these cells are irreversibly retained in the column. These results demonstrate the complexity of immunomagnetic cell separation and the need to further optimize the cell separation process.

The biology of human natural killer-cell subsets.

Human natural killer (NK) cells comprise approximately 15% of all circulating lymphocytes. Owing to their early production of cytokines and chemokines, and ability to lyse target cells without prior sensitization, NK cells are crucial components of the innate immune system. Human NK cells can be divided into two subsets based on their cell-surface density of CD56--CD56(bright) and CD56(dim)--each with distinct phenotypic properties. Now, there is ample evidence to suggest that these NK-cell subsets have unique functional attributes and, therefore, distinct roles in the human immune response. The CD56(dim) NK-cell subset is more naturally cytotoxic and expresses higher levels of Ig-like NK receptors and FCgamma receptor III (CD16) than the CD56(bright) NK-cell subset. By contrast, the CD56(bright) subset has the capacity to produce abundant cytokines following activation of monocytes, but has low natural cytotoxicity and is CD16(dim) or CD16(-). In addition, we will discuss other cell-surface receptors expressed differentially by human NK-cell subsets and the distinct functional properties of these subsets.

Human natural killer cells: a unique innate immunoregulatory role for the CD56(bright) subset.

During the innate immune response to infection, monocyte-derived cytokines (monokines), stimulate natural killer (NK) cells to produce immunoregulatory cytokines that are important to the host's early defense. Human NK cell subsets can be distinguished by CD56 surface density expression (ie, CD56(bright) and CD56(dim)). In this report, it is shown that CD56(bright) NK cells produce significantly greater levels of interferon-gamma, tumor necrosis factor-beta, granulocyte macrophage-colony-stimulating factor, IL-10, and IL-13 protein in response to monokine stimulation than do CD56(dim) NK cells, which produce negligible amounts of these cytokines. Further, qualitative differences in CD56(bright) NK-derived cytokines are shown to be dependent on the specific monokines present. For example, the monokine IL-15 appears to be required for type 2 cytokine production by CD56(bright) NK cells. It is proposed that human CD56(bright) NK cells have a unique functional role in the innate immune response as the primary source of NK cell-derived immunoregulatory cytokines, regulated in part by differential monokine production.

Fatal leukemia in interleukin-15 transgenic mice.

The role of inflammation in the early genesis of certain malignancies has recently been appreciated. Interleukin (IL)-15, a proinflammatory cytokine and growth factor, is required for lymphocyte homeostasis. Intriguingly, the expression of IL-15 protein is tightly controlled by multiple posttranscriptional mechanisms, suggesting that inappropriate expression of IL-15 may be detrimental to the host. We recently engineered a transgenic mouse in which the normal posttranscriptional control of IL-15 is eliminated, thereby overexpressing the murine IL-15 protein. IL-15 transgenic mice have early expansions in NK and CD8+ T lymphocytes and later develop fatal lymphocytic leukemia with a T-NK phenotype. This article recapitulates the phenotype of these IL-15 transgenic mice and discusses the utility of this model as a tool to further our understanding of leukemogenesis.

Interleukin-1beta costimulates interferon-gamma production by human natural killer cells.

Natural killer (NK) cells are an early source of immunoregulatory cytokines during the innate immune response to viruses, bacteria, and parasites. NK cells provide requisite IFN-gamma to monocytes for the elimination of obligate intracellular pathogens. IL-1beta is a pro-inflammatory cytokine produced by monocytes (i.e. a monokine) during the early immune response to infection, but its role in promoting human NK cell IFN-gamma production is unknown. The current study examines the ability of the monokine IL-1beta, plus IL-12, to costimulate IFN-gamma production by resting CD56(bright) and CD56(dim) human NK cell subsets. CD56(bright) NK cells stimulated with IL-1beta plus IL-12 produced abundant IFN-gamma protein, while little IFN-gamma was produced in identical cultures of CD56(dim) cells. In addition, upon activation with IL-1beta, CD56(bright) NK cells exhibited considerably greater phosphorylation of extracellular signal-regulated kinases p42/44 as compared to CD56(dim) NK cells. Quantitative PCR analysis showed brisk induction of IFN-gamma gene expression following costimulation with IL-1beta plus IL-12 in CD56(bright) NK cells, but intracellular flow cytometry revealed that only a fraction (42+/-2.3%) of CD56(bright) NK cells account for this high IFN-gamma production. These data suggest that the monokine IL-1beta is a potent costimulus of IFN-gamma production by a subset of NK cells following infectious insult.

Fatal leukemia in interleukin 15 transgenic mice follows early expansions in natural killer and memory phenotype CD8+ T cells.

Inflammation likely has a role in the early genesis of certain malignancies. Interleukin (IL)-15, a proinflammatory cytokine and growth factor, is required for lymphocyte homeostasis. Intriguingly, the expression of IL-15 protein is tightly controlled by multiple posttranscriptional mechanisms. Here, we engineered a transgenic mouse to overexpress IL-15 by eliminating these posttranscriptional checkpoints. IL-15 transgenic mice have early expansions in natural killer (NK) and CD8+ T lymphocytes. Later, these mice develop fatal lymphocytic leukemia with a T-NK phenotype. These data provide novel evidence that leukemia, like certain other cancers, can arise as the result of chronic stimulation by a proinflammatory cytokine.

Ontogeny and expansion of human natural killer cells: clinical implications.

Our knowledge of NK cells and their critical role in the innate immune system has increased enormously since their discovery several decades ago. However, it is only within the last 10 years that rational cytokine therapies, such as those utilizing low doses of IL-2, have been successful in expanding NK cells in patients with cancer and/or immunodeficiency. Such experiences in vivo have highlighted the importance of basing immunotherapeutic strategies on the known cellular and molecular properties of the targeted cell population. Recent advances in our understanding of the physiologic factors and events that orchestrate NK cell ontogeny, including IL-15 and receptor tyrosine kinase ligands to c-kit and flt3, provide novel therapeutic possibilities for cytokine therapy. This review summarizes our current understanding of human NK cell ontogeny, and links this knowledge to ongoing and future clinical strategies for the endogenous expansion of NK cells in patients with cancer and/or immunodeficiency.

Potential mechanisms of human natural killer cell expansion in vivo during low-dose IL-2 therapy.

The continuous, in vivo infusion of low-dose IL-2 selectively expands the absolute number of human natural killer (NK) cells after 4-6 weeks of therapy. The mechanism responsible for this expansion is unknown and was examined in this study. NK cells cultured at low concentrations of IL-2, comparable to those found during in vivo therapy, proliferate for 6 days and then exit the cell cycle. However, NK cells in vivo did not traverse the S/G(2)/M phase of the cell cycle during low-dose IL-2 therapy. Low concentrations of IL-2 delay programmed cell death of NK cells but have the same effect on resting T cells that do not expand in vivo. When CD34(+) bone marrow hematopoietic progenitor cells are cultured for 21 days with low concentrations of IL-2, they differentiate into CD56(+)CD3(-) NK cells, not T cells. Thus, the selective expansion of human NK cells during continuous in vivo infusion of low-dose IL-2 likely results from enhanced NK-cell differentiation from bone marrow progenitors, combined with an IL-2-dependent delay in NK-cell death, rather than proliferation of mature NK cells in the periphery.

Cytokine replacement in patients with HIV-1 non-Hodgkin's lymphoma: the rationale for low-dose interleukin-2 therapy.

PURPOSE: The drastic increase in the incidence of non-Hodgkin's lymphoma in patients infected with HIV-1 is testimony to the fact that our immune system is critical for the prevention of certain malignancies. Preclinical and clinical studies were conducted to (1) gain further insight into defects in immunity that can lead to malignant transformation and (2) determine if certain immune deficiencies could be corrected by cytokines delivered at doses that result in near-physiologic concentrations in vivo. METHODS: We have used the severe combined immune deficient mouse engrafted with human peripheral blood leukocytes from healthy individuals who are seropositive for the Epstein-Barr virus to study the spontaneous development of malignant Epstein-Barr virus-positive human B-cell lymphoproliferative disorder. RESULTS: We have demonstrated in this model that, in the absence of CD4+ T cells, cytokine replacement with low-dose interleukin (IL)-2 therapy can prevent Epstein-Barr virus-positive human B-cell lymphoproliferative disorder by interacting with mouse natural killer and human CD8+ T cells. We review our clinical experience with administration of low-dose IL-2 therapy in patients with HIV-1-related cancer, noting minimal toxicity and significant immune modulation. We provide evidence that this therapy can favorably alter the type 1 cytokine profile in vivo in these patients, and improve the cellular response to infectious insults in vitro. CONCLUSION: Early clinical studies with low-dose IL-2 therapy in patients with HIV-1-related lymphoma suggest that this therapy may have a role in the prevention and treatment of this disease.

Cutting edge: IL-15 costimulates the generalized Shwartzman reaction and innate immune IFN-gamma production in vivo.

Sequential administration of LPS to SCID mice results in the generalized Shwartzman reaction, manifesting as rapid mortality via cytokine-induced shock. Here we demonstrate that in vivo neutralization of IL-15 before LPS priming significantly reduced lethality in this reaction (p = 0.0172). We hypothesize that LPS priming induces IL-12 and IL-15 that costimulate NK cell-derived IFN-gamma. Such IFN-gamma may then in turn sensitize macrophages to elicit the Shwartzman reaction following a subsequent LPS challenge. Supporting this, IL-12 and IL-15 synergized to induce murine NK cell IFN-gamma production in vitro. LPS stimulation of SCID mouse splenocytes resulted in measurable IFN-gamma production, which was reduced when IL-15 was neutralized or IL-2/15Rbeta was blocked. Pretreatment with either anti-IL-2/15Rbeta or anti-IL-15 Abs reduced serum IFN-gamma protein following LPS administration to SCID mice. Collectively, these data provide the first in vivo evidence that IL-15 participates in LPS-induced innate immune IFN-gamma production and significantly contributes to the lethal Shwartzman reaction.

Differential cytokine and chemokine gene expression by human NK cells following activation with IL-18 or IL-15 in combination with IL-12: implications for the innate immune response.

NK cells constitutively express monocyte-derived cytokine (monokine) receptors and secrete cytokines and chemokines following monokine stimulation, and are therefore a critical component of the innate immune response to infection. Here we compared the effects of three monokines (IL-18, IL-15, and IL-12) on human NK cell cytokine and chemokine production. IL-18, IL-15, or IL-12 alone did not stimulate significant cytokine or chemokine production in resting NK cells. The combination of IL-18 and IL-12 induced extremely high amounts of IFN-gamma protein (225 +/- 52 ng/ml) and a 1393 +/- 643-fold increase in IFN-gamma gene expression over those in resting NK cells. IL-15 and IL-12 induced less IFN-gamma protein (24 +/- 10 ng/ml; p < 0.007) and only a 45 +/- 19-fold increase in IFN-gamma gene expression over those in resting NK cells. The CD56bright NK cell subset produced significantly more IFN-gamma following IL-18 and IL-12 compared with CD56dim NK cells (p < 0.008). However, the combination of IL-15 and IL-12 was significantly more potent than that of IL-18 and IL-12 for NK cell production of IL-10, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, and TNF-alpha at the protein and transcript levels. Granulocyte-macrophage CSF was optimally induced by IL-15 and IL-18. Resting CD56+ NK cells expressed IL-18R transcript that was up-regulated by IL-12 or IL-15. Our results show that distinct cytokine and chemokine patterns are induced in NK cells in response to different costimulatory signals from these three monokines. This suggests that NK cell cytokine production may be governed in part by the monokine milieu induced during the early proinflammatory response to infection and by the subset of NK cells present at the site of inflammation.

Natural killer cells from HIV-1+ patients produce C-C chemokines and inhibit HIV-1 infection.

Human NK cells have been shown to produce cytokines (e.g., IFN-gamma and TNF-alpha) and the chemokine macrophage inflammatory protein (MIP)-1alpha following stimulation with the combination of two monokines, IL-15 plus IL-12. The C-C chemokines MIP-1alpha, MIP-1beta, and RANTES have been identified as the major soluble macrophage-tropic HIV-1-suppressive factors produced by CD8+ T cells, which exert their action at the level of viral entry. Here, we demonstrate that monokine-activated NK cells, isolated from both normal and HIV-1+ donors, produce similar amounts of MIP-1alpha, MIP-1beta, and RANTES protein, in vitro. Further, supernatants of monokine-activated NK cells obtained from both normal donors and AIDS patients showed potent (routinely > or = 90%) suppressive activity against HIV-1 replication in vitro, compared with unstimulated control supernatants. NK cell supernatants inhibited both macrophage-tropic HIV-1(NFN-SX) and T cell-tropic HIV-1(NL4-3) replication in vitro, but not dual-tropic HIV-1(89.6). Importantly, the C-C chemokines MIP-1alpha, MIP-1beta, and RANTES were responsible only for a fraction of the HIV-1-suppressive activity exhibited by NK cell supernatants against macrophage-tropic HIV-1. Collectively these data indicate that NK cells from normal and HIV-1+ donors produce C-C chemokines and other unidentified factors that can inhibit both macrophage- and T cell-tropic HIV-1 replication in vitro. Since NK cells can be expanded in patients with HIV-1, AIDS, and AIDS malignancy in vivo, this cell type may have an important role in the in vivo regulation of HIV-1 infection.

Flt3 ligand promotes the generation of a distinct CD34(+) human natural killer cell progenitor that responds to interleukin-15.

Interleukin-15 (IL-15) is produced by human bone marrow (BM) stromal cells and can induce CD34(+) hematopoietic progenitor cells (HPCs) to differentiate into CD56(+)CD3(-) natural killer (NK) cells in the absence of stromal cells. IL-15 mediates its effects by signaling through the beta and gammac chains of the IL-2/15 receptor (R). The c-kit ligand (KL), also produced by stromal cells, enhances the expansion of NK cells from CD34(+) HPCs in the presence of IL-15, but alone has no ability to differentiate NK cells. Mice deficient in KL do not appear to have a quantitative deficiency in NK cells, suggesting that other stromal cell factors may contribute to NK cell expansion. Flt3 ligand (FL) is also produced by BM stromal cells and has homology with KL. Furthermore, mice with a targeted disruption of the FL gene have reduced numbers of NK cells. We evaluated here the effects of FL on human NK cell development and expansion from CD34(+) HPCs. Like KL, FL significantly enhanced the expansion of NK cells from CD34(+) HPCs in the presence of IL-15, compared with IL-15 alone. However, FL alone had no effect on NK cell differentiation. We therefore explored the mechanism by which FL promotes IL-15-mediated NK cell development. FL was found to induce IL-2/15Rbeta (CD122) expression on CD34(bright) HPCs. The CD34(bright) CD122(+) cell coexpressed CD38, but lacked expression of CD7, CD56, NK cell receptors (NKRs), or cytotoxic activity in the absence of IL-15. Using limiting dilution analysis in the presence of IL-15 alone, we demonstrated that the FL-induced CD34(bright)CD122(+) HPCs had an NK cell precursor frequency 20- to 60-fold higher than the CD34(dim/neg)CD122(-) HPCs and 65- to 235-fold higher than fresh CD34(+) HPCs. KL had similar effects as FL, but induced a significantly lower percentage of CD34(bright)CD122(+) cells (P