Megan A. Cooper

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.

Cytokine-induced memory-like natural killer cells

The mammalian immune response to infection is mediated by 2 broad arms, the innate and adaptive immune systems. Innate immune cells are a first-line defense against pathogens and are thought to respond consistently to infection, regardless of previous exposure, i.e., they do not exhibit memory of prior activation. By contrast, adaptive immune cells display immunologic memory that has 2 basic characteristics, antigen specificity and an amplified response upon subsequent exposure. Whereas adaptive immune cells have rearranged receptor genes to recognize the universe of antigens, natural killer (NK) cells are innate immune lymphocytes with a limited repertoire of germ-line encoded receptors for target recognition. NK cells also produce cytokines such as IFN-gamma (IFN-γ) to protect the host during the innate response to infection. Herein, we show that cytokine-activated NK cells transferred into naïve hosts can be specifically detected 7–22 days later when they are phenotypically similar to naïve cells and are not constitutively producing IFN-γ. However, they produce significantly more IFN-γ when restimulated. This memory-like property is intrinsic to the NK cell. By contrast, memory-like NK cells do not express granzyme B protein and kill targets similarly to naïve NK cells. Thus, these experiments identify an ability of innate immune cells to retain an intrinsic memory of prior activation, a function until now attributed only to antigen-specific adaptive immune cells.

Interleukin-2 and interleukin-15: immunotherapy for cancer.

Interleukin (IL)-2 and IL-15 are two cytokine growth factors that regulate lymphocyte function and homeostasis. Early clinical interest in the use of IL-2 in the immunotherapy of renal cell carcinoma and malignant melanoma demonstrated the first efficacy for cytokine monotherapy in the treatment of neoplastic disease. Advances in our understanding of the cellular and molecular biology of IL-2 and its receptor complex have provided rationale to better utilize IL-2 to expand and activate immune effectors in patients with cancer. Exciting new developments in monoclonal antibodies recognizing tumor targets and tumor vaccines have provided new avenues to combine with IL-2 therapy in cancer patients. IL-15, initially thought to mediate similar biological effects as IL-2, has been shown to have unique properties in basic and pre-clinical studies that may be of benefit in the immunotherapy of cancer. This review first summarizes the differences between IL-2 and IL-15 and highlights that better understanding of normal physiology creates new ideas for the immunotherapy of cancer. The application of high, intermediate, and low/ultra low dose IL-2 therapy in clinical trials of cancer patients is discussed, along with new avenues for its use in neoplastic diseases. The growing basic and pre-clinical evidence demonstrating that IL-15 may be useful in immunotherapy approaches to cancer is also presented.

Cytokine activation induces human memory-like NK cells

Natural killer (NK) cells are lymphocytes that play an important role in the immune response to infection and malignancy. Recent studies in mice have shown that stimulation of NK cells with cytokines or in the context of a viral infection results in memory-like properties. We hypothesized that human NK cells exhibit such memory-like properties with an enhanced recall response after cytokine preactivation. In the present study, we show that human NK cells preactivated briefly with cytokine combinations including IL-12, IL-15, and IL-18 followed by a 7- to 21-day rest have enhanced IFN-γ production after restimulation with IL-12 + IL-15, IL-12 + IL-18, or K562 leukemia cells. This memory-like phenotype was retained in proliferating NK cells. In CD56(dim) NK cells, the memory-like IFN-γ response was correlated with the expression of CD94, NKG2A, NKG2C, and CD69 and a lack of CD57 and KIR. Therefore, human NK cells have functional memory-like properties after cytokine activation, which provides a novel rationale for integrating preactivation with combinations of IL-12, IL-15, and IL-18 into NK cell immunotherapy strategies.

Early-onset lymphoproliferation and autoimmunity caused by germline STAT3 gain-of-function mutations.

Germline loss-of-function mutations in the transcription factor signal transducer and activator of transcription 3 (STAT3) cause immunodeficiency, whereas somatic gain-of-function mutations in STAT3 are associated with large granular lymphocytic leukemic, myelodysplastic syndrome, and aplastic anemia. Recently, germline mutations in STAT3 have also been associated with autoimmune disease. Here, we report on 13 individuals from 10 families with lymphoproliferation and early-onset solid-organ autoimmunity associated with 9 different germline heterozygous mutations in STAT3. Patients exhibited a variety of clinical features, with most having lymphadenopathy, autoimmune cytopenias, multiorgan autoimmunity (lung, gastrointestinal, hepatic, and/or endocrine dysfunction), infections, and short stature. Functional analyses demonstrate that these mutations confer a gain-of-function in STAT3 leading to secondary defects in STAT5 and STAT1 phosphorylation and the regulatory T-cell compartment. Treatment targeting a cytokine pathway that signals through STAT3 led to clinical improvement in 1 patient, suggesting a potential therapeutic option for such patients. These results suggest that there is a broad range of autoimmunity caused by germline STAT3 gain-of-function mutations, and that hematologic autoimmunity is a major component of this newly described disorder. Some patients for this study were enrolled in a trial registered at www.clinicaltrials.gov as #NCT00001350.

Cytokine-induced memory-like natural killer cells exhibit enhanced responses against myeloid leukemia

Cytokine-induced differentiation of memory-like natural killer cells enhances antileukemia responses. Natural killers of leukemia Natural killer cells, part of the innate immune system, play a role in immune responses against exogenous pathogens as well as cancer. Recent studies have identified the existence of memory-like characteristics in some natural killer cells, and Romee et al. investigated these memory cells’ potential as a cancer therapy. The authors compared human natural killer memory cells to non-memory control cells, then demonstrated their effectiveness against myeloid leukemia models in vitro and in mice. They also performed a clinical trial in human patients with acute myeloid leukemia, where the natural killer memory-like cells again demonstrated antileukemia effects, some of which produced clinical remissions. Natural killer (NK) cells are an emerging cellular immunotherapy for patients with acute myeloid leukemia (AML); however, the best approach to maximize NK cell antileukemia potential is unclear. Cytokine-induced memory-like NK cells differentiate after a brief preactivation with interleukin-12 (IL-12), IL-15, and IL-18 and exhibit enhanced responses to cytokine or activating receptor restimulation for weeks to months after preactivation. We hypothesized that memory-like NK cells exhibit enhanced antileukemia functionality. We demonstrated that human memory-like NK cells have enhanced interferon-γ production and cytotoxicity against leukemia cell lines or primary human AML blasts in vitro. Using mass cytometry, we found that memory-like NK cell functional responses were triggered against primary AML blasts, regardless of killer cell immunoglobulin-like receptor (KIR) to KIR-ligand interactions. In addition, multidimensional analyses identified distinct phenotypes of control and memory-like NK cells from the same individuals. Human memory-like NK cells xenografted into mice substantially reduced AML burden in vivo and improved overall survival. In the context of a first-in-human phase 1 clinical trial, adoptively transferred memory-like NK cells proliferated and expanded in AML patients and demonstrated robust responses against leukemia targets. Clinical responses were observed in five of nine evaluable patients, including four complete remissions. Thus, harnessing cytokine-induced memory-like NK cell responses represents a promising translational immunotherapy approach for patients with AML.

Hidden talents of natural killers: NK cells in innate and adaptive immunity

Natural killer (NK) cells are innate immune lymphocytes capable of killing target cells and producing immunoregulatory cytokines. Herein, we discuss recent studies that indicate that NK cells span the conventional boundaries between innate and adaptive immunity. For example, it was recently discovered that NK cells have the capacity for memory‐like responses, a property that was previously thought to be limited to adaptive immunity. NK cells have also been identified in multiple tissues, and a subset of cells that specialize in the production of the TH17 cytokine IL‐22, NK‐22s, was recently described in mucosal‐associated lymphoid tissue. Finally, we review work that shows that NK cells develop at sites that were traditionally thought to be occupied only by adaptive immune cells, including the thymus and lymph nodes.

Preactivation with IL-12, IL-15, and IL-18 induces CD25 and a functional high-affinity IL-2 receptor on human cytokine-induced memory-like natural killer cells.

Natural killer (NK) cells are effector lymphocytes that are under clinical investigation for the adoptive immunotherapy of hematologic malignancies, especially acute myeloid leukemia. Recent work in mice has identified innate memory-like properties of NK cells. Human NK cells also exhibit memory-like properties, and cytokine-induced memory-like (CIML) NK cells are generated via brief preactivation with IL-12, IL-15, and IL-18, which later exhibit enhanced functionality upon restimulation. However, the optimal cytokine receptors and signals for maintenance of enhanced function and homeostasis after preactivation remain unclear. Here, we show that IL-12, IL-15, and IL-18 preactivation induces a rapid and prolonged expression of CD25, resulting in a functional high-affinity IL-2 receptor (IL-2Rαβγ) that confers responsiveness to picomolar concentrations of IL-2. The expression of CD25 correlated with STAT5 phosphorylation in response to picomolar concentrations of IL-2, indicating the presence of a signal-competent IL-2Rαβγ. Furthermore, picomolar concentrations of IL-2 acted synergistically with IL-12 to costimulate IFN-γ production by preactivated NK cells, an effect that was CD25 dependent. Picomolar concentrations of IL-2 also enhanced NK cell proliferation and cytotoxicity via the IL-2Rαβγ. Further, after adoptive transfer into immunodeficient NOD-SCID-γc(-/-) mice, human cytokine-preactivated NK cells expand preferentially in response to exogenous IL-2. Collectively, these data demonstrate that human CIML NK cells respond to IL-2 via IL-2Rαβγ with enhanced survival and functionality, and they provide additional rationale for immunotherapeutic strategies that include brief cytokine preactivation before adoptive NK cell transfer, followed by low-dose IL-2 therapy.

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.

Interleukin-1β costimulates interferon-γ 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‐γ to monocytes for the elimination of obligate intracellular pathogens. IL‐1β 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‐γ production is unknown. The current study examines the ability of the monokine IL‐1β, plus IL‐12, to costimulate IFN‐γ production by resting CD56bright and CD56dim human NK cell subsets. CD56bright NK cells stimulated with IL‐1β plus IL‐12 produced abundant IFN‐γ protein, while little IFN‐γ was produced in identical cultures of CD56dim cells. In addition, upon activation with IL‐1β, CD56bright NK cells exhibited considerably greater phosphorylation of extracellular signal‐regulated kinases p42/44 as compared to CD56dim NK cells. Quantitative PCR analysis showed brisk induction of IFN‐γ gene expression following costimulation with IL‐1β plus IL‐12 in CD56bright NK cells, but intracellular flow cytometry revealed that only a fraction (42±2.3%) of CD56bright NK cells account for this high IFN‐γ production. These data suggest that the monokine IL‐1β is a potent costimulus of IFN‐γ production by a subset of NK cells following infectious insult.