GVHD-associated, inflammasome-mediated loss of function in adoptively transferred myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSCs) are a naturally occurring immune regulatory population associated with inhibition of ongoing inflammatory responses. In vitro generation of MDSCs from bone marrow has been shown to enhance survival in an acute model of lethal graft-versus-host disease (GVHD). However, donor MDSC infusion only partially ameliorates GVHD lethality. In order to improve the potential therapeutic benefit and ultimately survival outcomes, we set out to investigate the fate of MDSCs after transfer in the setting of acute GVHD (aGVHD). MDSCs transferred to lethally irradiated recipients of allogeneic donor hematopoietic grafts are exposed to an intense inflammatory environment associated with aGVHD, which we now show directly undermines their suppressive capacity. Under a conditioning regimen and GVHD inflammatory settings, MDSCs rapidly lose suppressor function and their potential to inhibit GVHD lethality, which is associated with their induced conversion toward a mature inflammasome-activated state. We find even brief in vitro exposure to inflammasome-activating mediators negates the suppressive potential of cultured murine and human-derived MDSCs. Consistent with a role for the inflammasome, donor MDSCs deficient in the adaptor ASC (apoptosis-associated speck-like protein containing a CARD), which assembles inflammasome complexes, conferred improved survival of mice developing GVHD compared with wild-type donor MDSCs. These data suggest the use of MDSCs as a therapeutic approach for preventing GVHD and other systemic inflammatory conditions will be more effective when combined with approaches limiting in vivo MDSC inflammasome activation, empowering MDSCs to maintain their suppressive potential.

Induced Pluripotent Stem Cell-Derived Natural Killer Cells for Treatment of Ovarian Cancer.

Natural killer (NK) cells can provide effective immunotherapy for ovarian cancer. Here, we evaluated the ability of NK cells isolated from peripheral blood (PB) and NK cells derived from induced pluripotent stem cell (iPSC) to mediate killing of ovarian cancer cells in a mouse xenograft model. A mouse xenograft model was used to evaluate the intraperitoneal delivery of three different NK cell populations: iPSC-derived NK cells, PB-NK cells that had been activated and expanded in long-term culture, and overnight activated PB-NK cells that were isolated through CD3/CD19 depletion of PB B and T cells. Bioluminescent imaging was used to monitor tumor burden of luciferase expressing tumor lines. Tumors were allowed to establish prior to administering NK cells via intraperitoneal injection. These studies demonstrate a single dose of any of the three NK cell populations significantly reduced tumor burden. When mice were given three doses of either iPSC-NK cells or expanded PB-NK cells, the median survival improved from 73 days in mice untreated to 98 and 97 days for treated mice, respectively. From these studies, we conclude iPSC-derived NK cells mediate antiovarian cancer killing at least as well as PB-NK cells, making these cells a viable resource for immunotherapy for ovarian cancer. Due to their ability to be easily differentiated into NK cells and their long-term expansion potential, iPSCs can be used to produce large numbers of well-defined NK cells that can be banked and used to treat a large number of patients including treatment with multiple doses if necessary.

UNC-45A Is a Nonmuscle Myosin IIA Chaperone Required for NK Cell Cytotoxicity via Control of Lytic Granule Secretion.

NK cell's killing is a tightly regulated process under the control of specific cytoskeletal proteins. This includes Wiskott-Aldrich syndrome protein, Wiskott-Aldrich syndrome protein-interacting protein, cofilin, Munc13-4, and nonmuscle myosin IIA (NMIIA). These proteins play a key role in controlling NK-mediated cytotoxicity either via regulating the attachment of lytic granules to the actin-based cytoskeleton or via promoting the cytoskeletal reorganization that is requisite for lytic granule release. UNC-45A is a highly conserved member of the UNC-45/CRO1/She4p family of proteins that act as chaperones for both conventional and nonconventional myosin. Although we and others have shown that in lower organisms and in mammalian cells NMIIA-associated functions, such as cytokinesis, cell motility, and organelle trafficking, are dependent upon the presence of UNC-45A, its role in NK-mediated functions is largely unknown. In this article, we describe UNC-45A as a key regulator of NK-mediated cell toxicity. Specifically we show that, in human NK cells, UNC-45A localize at the NK cell immunological synapse of activated NK cells and is part of the multiprotein complex formed during NK cell activation. Furthermore, we show that UNC-45A is disposable for NK cell immunological synapse formation and lytic granules reorientation but crucial for lytic granule exocytosis. Lastly, loss of UNC-45A leads to reduced NMIIA binding to actin, suggesting that UNC-45A is a crucial component in regulating human NK cell cytoskeletal dynamics via promoting the formation of actomyosin complexes.

Expansion and homing of adoptively transferred human natural killer cells in immunodeficient mice varies with product preparation and in vivo cytokine administration: implications for clinical therapy.

Natural killer (NK) cell efficacy correlates with in vivo proliferation, and we hypothesize that NK cell product manipulations may optimize this endpoint. Xenotransplantation was used to compare good manufacturing practice (GMP) grade freshly activated NK cells (FA-NK) and ex vivo expanded NK cells (Ex-NK). Cells were infused into NOD scid IL2 receptor gamma chain knockout (NSG) mice followed by IL-2, IL-15, or no cytokines. Evaluation of blood, spleen, and marrow showed that persistence and expansion was cytokine dependent, IL-15 being superior to IL-2. Cryopreservation and immediate infusion resulted in less cytotoxicity and fewer NK cells in vivo, and this could be rescued in FA-NK by overnight culture and testing the next day. Marked differences in the kinetics and homing of FA-NK versus Ex-NK were apparent: FA-NK cells preferentially homed to spleen and persisted longer after cytokine withdrawal. These data suggest that cryopreservation of FA-NK and Ex-NK is detrimental and that culture conditions profoundly affect homing, persistence, and expansion of NK cells in vivo. The NSG mouse model is an adjuvant to in vitro assays before clinical testing.

Tim-3 is an inducible human natural killer cell receptor that enhances interferon gamma production in response to galectin-9.

NK-cell function is regulated by the integration of signals received from activating and inhibitory receptors. Here we show that a novel immune receptor, T-cell Ig and mucin-containing domain-3 (Tim-3), is expressed on resting human NK cells and is up-regulated on activation. The NK92 NK-cell line engineered to overexpress Tim-3 showed a marked increase in IFN-γ production in the presence of soluble rhGal-9 or Raji tumor cells engineered to express Gal-9. The Tim-3(+) population of low-dose IL-12/IL-18-activated primary NK cells significantly increased IFN-γ production in response to soluble rhGal-9, Gal-9 presented by cell lines, and primary acute myelogenous leukemia (AML) targets that endogenously express Gal-9. This effect is highly specific as Tim-3 Ab blockade significantly decreased IFN-γ production, and Tim-3 cross-linking induced ERK activation and degradation of IκBα. Exposure to Gal-9-expressing target cells had little effect on CD107a degranulation. Reconstituted NK cells obtained from patients after hematopoietic cell transplantation had diminished expression of Tim-3 compared with paired donors. This observation correlates with the known IFN-γ defect seen early posttransplantation. In conclusion, we show that Tim-3 functions as a human NK-cell coreceptor to enhance IFN-γ production, which has important implications for control of infectious disease and cancer.

Intraperitoneal delivery of human natural killer cells for treatment of ovarian cancer in a mouse xenograft model.

BACKGROUND AIMS: There is an urgent need for novel therapeutic strategies for relapsed ovarian cancer. Dramatic clinical anti-tumor effects have been observed with interleukin (IL)-2 activated natural killer (NK) cells; however, intravenous delivery of NK cells in patients with ovarian cancer has not been successful in ameliorating disease. We investigated in vivo engraftment of intraperitoneally (IP) delivered NK cells in an ovarian cancer xenograft model to determine if delivery mode can affect tumor cell killing and circumvent lack of NK cell expansion. METHODS: An ovarian cancer xenograft mouse model was established to evaluate efficacy of IP-delivered NK cells. Tumor burden was monitored by bioluminescent imaging of luciferase-expressing ovarian cancer cells. NK cell persistence, tumor burden and NK cell trafficking were evaluated. Transplanted NK cells were evaluated by flow cytometry and cytotoxicity assays. RESULTS: IP delivery of human NK cells plus cytokines led to high levels of circulating NK and was effective in clearing intraperitoneal ovarian cancer burden in xenografted mice. NK cells remained within the peritoneal cavity 54 days after injection and had markers of maturation. Additionally, surviving NK cells were able to kill ovarian cancer cells at a rate similar to pre-infusion levels, supporting that in vivo functionality of human NK cells can be maintained after IP infusion. CONCLUSIONS: IP delivery of NK cells leads to stable engraftment and antitumor response in an ovarian cancer xenograft model. These data support further pre-clinical and clinical evaluation of IP delivery of allogeneic NK cells in ovarian cancer.

Cutting edge: microRNA-181 promotes human NK cell development by regulating Notch signaling.

MicroRNAs (miRs) have recently been identified as important regulators of gene expression at the posttranscriptional level. Although it has clearly been established that miRs influence the ontogeny of several immune cell lineages, the role of individual miRs during NK cell development has not been described. In this study, we show that miR-181 expression levels have a profound impact on the development of human NK cells from CD34(+) hematopoietic progenitor cells and IFN-γ production in primary CD56(+) NK cells. We also demonstrate that nemo-like kinase (NLK), an inhibitor of Notch signaling, is a target of miR-181 in NK cells, and knockdown of NLK mirrors the developmental effect of miR-181 overexpression. We conclude that miR-181 promotes NK cell development, at least in part, through the suppression of NLK, providing an important link between miRs and Notch signaling.

The transcription factor c-Myc enhances KIR gene transcription through direct binding to an upstream distal promoter element.

The killer cell immunoglobulin-like receptor (KIR) repertoire of natural killer (NK) cells determines their ability to detect infected or transformed target cells. Although epigenetic mechanisms play a role in KIR gene expression, work in the mouse suggests that other regulatory elements may be involved at specific stages of NK-cell development. Here we report the effects of the transcription factor c-Myc on KIR expression. c-Myc directly binds to, and promotes transcription from, a distal element identified upstream of most KIR genes. Binding of endogenous c-Myc to the distal promoter element is significantly enhanced upon interleukin-15 (IL-15) stimulation in peripheral blood NK cells and correlates with an increase in KIR transcription. In addition, the overexpression of c-Myc during NK-cell development promotes transcription from the distal promoter element and contributes to the overall transcription of multiple KIR genes. Our data demonstrate the significance of the 5' promoter element upstream of the conventional KIR promoter region and support a model whereby IL-15 stimulates c-Myc binding at the distal KIR promoter during NK-cell development to promote KIR transcription. This finding provides a direct link between NK-cell activation signals and KIR expression required for acquisition of effector function during NK-cell education.

Natural killer cell killing of acute myelogenous leukemia and acute lymphoblastic leukemia blasts by killer cell immunoglobulin-like receptor-negative natural killer cells after NKG2A and LIR-1 blockade.

Although the study of natural killer (NK) cell alloreactivity has been dominated by studies of killer cell immunoglobulin-like receptors (KIRs), we hypothesized that NKG2A and LIR-1, present on 53% +/- 13% and 36% +/- 18% of normal NK cells, respectively, play roles in the NK cell killing of primary leukemia targets. KIR(-) cells, which compose nearly half of the circulating NK cell population, exhibit tolerance to primary leukemia targets, suggesting signaling through other inhibitory receptors. Both acute myelogenous leukemia and acute lymphoblastic leukemia targets were rendered susceptible to lysis by fresh resting KIR(-) NK cells when inhibitory receptor-major histocompatibility class I interactions were blocked by pan-HLA antibodies, demonstrating that these cells are functionally competent. Blockade of a single inhibitory receptor resulted in slightly increased killing, whereas combined LIR-1 and NKG2A blockade consistently resulted in increased NK cell cytotoxicity. Dual blockade of NKG2A and LIR-1 led to significant killing of targets by resting KIR(-) NK cells, demonstrating that this population is not hyporesponsive. Together these results suggest that alloreactivity of a significant fraction of KIR(-) NK cells is mediated by NKG2A and LIR-1. Thus strategies to interrupt NKG2A and LIR-1 in combination with anti-KIR blockade hold promise for exploiting NK cell therapy in acute leukemias.

Activated notch supports development of cytokine producing NK cells which are hyporesponsive and fail to acquire NK cell effector functions.

Natural Killer (NK) cells are powerful effectors of cytotoxicity against "stressed" cells. They also produce cytokines and chemokines to activate the adaptive immune response. Understanding NK cell development and maturation may have implications for cancer therapy and for immunity against infections. We hypothesized that Notch signaling, critical for hematopoesis, would be involved in NK cell development. The role of constitutively activated Notch1 (ICN) on NK cell maturation was studied using human umbilical cord blood (UCB) progenitors cultured on a murine embryonic liver stroma cell line (EL08-1D2) and human cytokines. UCB CD34(+)/ICN(+) sorted cells resulted in a population of CD7(+) early lymphoid precursors and subsequent NK lineage commitment independent of stroma or IL-15. Early expression of L-selectin on ICN(+) precursors suggested their homing competence. These precursors further committed to the NK lineage, and were capable of producing cytokines and chemokines such as interleukin (IL)-13, granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), yet poorly acquired NK inhibitory receptors and cytotoxic effector function. In the presence of stroma, ICN(+) precursors also gave rise to a population of early T lineage committed cells characterized by expression of cytoplasmic CD3 gamma, epsilon, and delta chains, RAG1/2, and production of IL-2, suggesting bona fide Th1 commitment. Importantly, signals from EL08-1D2 stroma were required for this development process. In conclusion, sustained Notch signaling can replace stroma in differentiation of a common CD7(+) lymphoid precursor from UCB CD34(+) progenitors and induce NK cell commitment. However, these NK cells are immature in their cytokine production profile, are hyporesponsive, and poorly acquire NK cell receptors involved in self-tolerance and effector function.

Mouse fetal and embryonic liver cells differentiate human umbilical cord blood progenitors into CD56-negative natural killer cell precursors in the absence of interleukin-15.

OBJECTIVE: Human natural killer (NK) cell maturation involves the orderly acquisition of NK cell receptors. Our aim was to understand how stromal interactions and cytokines are important in this developmental process. MATERIALS AND METHODS: Human umbilical cord blood (UCB) CD34(+)/Lin(-)/CD38(-) cells were cultured on two murine stromal cell lines (AFT024 and EL08-1D2) in a switch culture to study NK cell development. RESULTS: When human progenitors were cultured on AFT024 with interleukin (IL)-3 and Flt3 ligand (Flt3L) in the absence of interleukin (IL)-15, NK cell differentiation occurred, albeit at low frequency. These conditions favored the accumulation of CD56(-) NK cell precursors (CD34(+)CD7(-), CD34(+)CD7(+), and CD34(-)CD7(+) cells), which are populations rare in adult blood but abundant in fresh UCB. In secondary culture, addition of IL-3 or IL-3 + Flt3L to IL-15 increased the absolute number of CD56(+) NK cells from precursors and the acquisition of CD94 and killer immunoglobulin-like receptors (KIR). To further explore the microenvironment in early NK cell maturation, a cell line derived from murine embryonic liver (EL08-1D2) was studied. NK cell development and KIR acquisition was superior with EL08-1D2, which supported the differentiation of NK cell precursors, NK cell commitment, and proliferation. CONCLUSION: Although the earliest events in NK cell maturation do not require exogenous human IL-15, it is required at a later stage of NK cell commitment. At a minimum, murine stroma, IL-3, and Flt3L are required to recapitulate early NK cell development and differentiation into distinct NK cell precursors. EL08-1D2 induces KIR acquisition suggesting that extrinsic signals in NK cell development are conserved between mouse and man.

GSK3 Inhibition Drives Maturation of NK Cells and Enhances Their Antitumor Activity.

Maturation of human natural killer (NK) cells as defined by accumulation of cell-surface expression of CD57 is associated with increased cytotoxic character and TNF and IFNγ production upon target-cell recognition. Notably, multiple studies point to a unique role for CD57+ NK cells in cancer immunosurveillance, yet there is scant information about how they mature. In this study, we show that pharmacologic inhibition of GSK3 kinase in peripheral blood NK cells expanded ex vivo with IL15 greatly enhances CD57 upregulation and late-stage maturation. GSK3 inhibition elevated the expression of several transcription factors associated with late-stage NK-cell maturation including T-BET, ZEB2, and BLIMP-1 without affecting viability or proliferation. When exposed to human cancer cells, NK cell expanded ex vivo in the presence of a GSK3 inhibitor exhibited significantly higher production of TNF and IFNγ, elevated natural cytotoxicity, and increased antibody-dependent cellular cytotoxicity. In an established mouse xenograft model of ovarian cancer, adoptive transfer of NK cells conditioned in the same way also displayed more robust and durable tumor control. Our findings show how GSK3 kinase inhibition can greatly enhance the mature character of NK cells most desired for effective cancer immunotherapy. Cancer Res; 77(20); 5664-75. ©2017 AACR.

IL15 Trispecific Killer Engagers (TriKE) Make Natural Killer Cells Specific to CD33+ Targets While Also Inducing Persistence, In Vivo Expansion, and Enhanced Function.

PURPOSE: The effectiveness of NK cell infusions to induce leukemic remission is limited by lack of both antigen specificity and in vivo expansion. To address the first issue, we previously generated a bispecific killer engager (BiKE) containing single-chain scFv against CD16 and CD33 to create an immunologic synapse between NK cells and CD33(+) myeloid targets. We have now incorporated a novel modified human IL15 crosslinker, producing a 161533 trispecific killer engager (TriKE) to induce expansion, priming, and survival, which we hypothesize will enhance clinical efficacy. EXPERIMENTAL DESIGN: Reagents were tested in proliferation and functional assays and in an in vivo xenograft model of AML. RESULTS: When compared with the 1633 BiKE, the 161533 TriKE induced superior NK cell cytotoxicity, degranulation, and cytokine production against CD33(+) HL-60 targets and increased NK survival and proliferation. Specificity was shown by the ability of a 1615EpCAM TriKE to kill CD33-EpCAM(+) targets. Using NK cells from patients after allogeneic stem cell transplantation when NK cell function is defective, the 161533 TriKE restored potent NK function against primary AML targets and induced specific NK cell proliferation. These results were confirmed in an immunodeficient mouse HL-60-Luc tumor model where the 161533 TriKE exhibited superior antitumor activity and induced in vivo persistence and survival of human NK cells for at least 3 weeks. CONCLUSIONS: Off-the-shelf 161533 TriKE imparts antigen specificity and promotes in vivo persistence, activation, and survival of NK cells. These qualities are ideal for NK cell therapy of myeloid malignancies or targeting antigens of solid tumors. Clin Cancer Res; 22(14); 3440-50. ©2016 AACRSee related commentary by Talmadge, p. 3419.

Isolation and characterization of canine natural killer cells.

NK cells are non-T, non-B lymphocytes that kill target cells without previous activation. The immunophenotype and function of these cells in humans and mice are well defined, but canine NK cells remain incompletely characterized. Our objectives were to isolate and culture canine peripheral blood NK cells, and to define their immunophenotype and killing capability. PBMC were obtained from healthy dogs and T cells were depleted by immunomagnetic separation. The residual cells were cultured in media supplemented with IL-2, IL-15 or both, or with mouse embryonic liver (EL) feeder cells. Non-T, non-B lymphocytes survived and expanded in these cultures. IL-2 was necessary and sufficient for survival; the addition of IL-15 was necessary for expansion, but IL-15 alone did not support survival. Culture with EL cells and IL-2 also fostered survival and expansion. The non-T, non-B lymphocytes uniformly expressed CD45, MHC I, and showed significant cytotoxic activity against CTAC targets. Expression of MHC II, CD11/18 was restricted to subsets of these cells. The data show that cells meeting the criteria for NK cells in other species, i.e., non-T, non-B lymphocytes with cytotoxic activity, can be expanded from canine PBMC by T-cell depletion and culture with cytokines or feeder cells.

Heterodimeric bispecific single-chain variable-fragment antibodies against EpCAM and CD16 induce effective antibody-dependent cellular cytotoxicity against human carcinoma cells.

A heterodimeric bispecific biological recombinant drug was synthesized by splicing DNA fragments from two fully humanized single-chain variable-fragment (scFV) antibody fragments forming a novel drug simultaneously recognizing the CD16 natural killer (NK) cell marker and the cancer marker epithelial cell adhesion molecule (EpCAM). The drug precipitously enhanced the killing of human carcinomas of the prostate, breast, colon, head, and neck even at very low effector:target ratios. The drug EpCAM16 rendered even nonactivated NK cell-proficient killers and activated them to kill via degranulation and cytokine production. Studies show that bispecific antibodies can be used to induce proficient killing of the carcinoma targets that ordinarily are resistant to NK-mediated killing. Apparently, the innate immune system can be effectively recruited to kill cancer cells using the bispecific antibody platform and EpCAM targeting.

Bispecific and trispecific killer cell engagers directly activate human NK cells through CD16 signaling and induce cytotoxicity and cytokine production.

This study evaluates the mechanism by which bispecific and trispecific killer cell engagers (BiKEs and TriKEs) act to trigger human natural killer (NK) cell effector function and investigates their ability to induce NK cell cytokine and chemokine production against human B-cell leukemia. We examined the ability of BiKEs and TriKEs to trigger NK cell activation through direct CD16 signaling, measuring intracellular Ca²âº mobilization, secretion of lytic granules, induction of target cell apoptosis, and production of cytokine and chemokines in response to the Raji cell line and primary leukemia targets. Resting NK cells triggered by the recombinant reagents led to intracellular Ca²âº mobilization through direct CD16 signaling. Coculture of reagent-treated resting NK cells with Raji targets resulted in significant increases in NK cell degranulation and target cell death. BiKEs and TriKEs effectively mediated NK cytotoxicity of Raji targets at high and low effector-to-target ratios and maintained functional stability after 24 and 48 hours of culture in human serum. NK cell production of IFN-γ, TNF-α, granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-8, macrophage inflammatory protein (MIP)-1α, and regulated and normal T cell expressed and secreted (RANTES) was differentially induced in the presence of recombinant reagents and Raji targets. Moreover, significant increases in NK cell degranulation and enhancement of IFN-γ production against primary acute lymphoblastic leukemia and chronic lymphocytic leukemia targets were induced with reagent treatment of resting NK cells. In conclusion, BiKEs and TriKEs directly trigger NK cell activation through CD16, significantly increasing NK cell cytolytic activity and cytokine production against tumor targets, showing their therapeutic potential for enhancing NK cell immunotherapies for leukemias and lymphomas.

iTRAQ is a useful method to screen for membrane-bound proteins differentially expressed in human natural killer cell types.

We are interested in the biological as well as the molecular processes involved in natural killer (NK) cell development and function. Determining the proteomic complement could be a useful tool in predicting cellular function and fate. For the first time shown here, we have utilized iTRAQ, a new method that allows identification and quantification of proteins between multiple samples, to determine the expression of membrane-bound proteins in two previously characterized human NK cell populations. One population was derived from umbilical cord blood (UCB) stem cells (CD34+38-Lin-) and the other from expanded CD3-depleted adult peripheral blood. iTRAQ was employed for multiplex peptide labeling of proteins from fractionated membranes followed by two-dimensional high-performance liquid chromatography (2D-HPLC), and tandem mass spectrometry was used to identify protein signatures. We were able to identify and quantify differences in expression levels of 400-800 proteins in a typical experiment. Ontology analysis showed the majority of the proteins to be involved in cell signaling, nucleic acid binding, or mitochondrial function. Nearly all proteins were associated with the plasma membrane, membrane-bound organelle (lysosome or mitochondria), or nucleus. We found several novel proteins highly expressed in UCB stem cell derived NK cells compared to adult NK cells including CD9, alpha-2 macroglobulin, brain abundant signaling protein (BASP1), and allograft inflammatory factor-1 (AIF-1). In addition, we were able to confirm several of our iTRAQ results by RT-PCR, Western blot, and fluorescence-activated cell-sorting (FACS) analysis. This is the first demonstration and verification using iTRAQ to screen for membrane-bound protein differences in human NK cells and represents a powerful new tool in the field of proteomics.

The unexpected effect of cyclosporin A on CD56+CD16- and CD56+CD16+ natural killer cell subpopulations.

Cyclosporin A (CSA) is commonly used to prevent graft-versus-host disease. The influence of CSA on T-cell function has been extensively investigated; however, the effect of CSA on natural killer (NK) cells is less understood. NK cells were cultured with IL-2 and IL-15 with and without CSA for 1 week. Compared with controls, CSA-treated cultures showed fewer CD56(+)CD16(+)KIR(+) NK cells and a reciprocal increase in CD56(+)CD16(-)KIR(-) cells. These changes were due mainly to a reduced proliferation of the CD56(dim) NK-cell subpopulation and a relative resistance of CD56(bright) NK cells to CSA. Following coculture with K562 targets, CSA-exposed NK cells differed from controls and lacked Ca(2+) oscillations, nuclear factor of activated T cells (NFAT) dephosphorylation, and NFAT nuclear translocation. NK cells cultured in CSA retained cytotoxicity against K562, Raji, and KIR ligand-expressing lymphoblastoid cells. NK cells cultured in CSA showed increases in NKp30 and reductions in NKp44 and NKG2D. Following IL-12 and IL-18 stimulation, CSA-treated NK cells showed more IFN-gamma-producing cells. Using in vitro NK-cell differentiation, progenitor cells gave rise to more CD56(+)KIR(-) NK cells in the presence of CSA than controls. Collectively, these studies show that CSA influences NK-cell function and phenotype, which may have important implications for graft-versus-leukemia effects.

A subpopulation of human peripheral blood NK cells that lacks inhibitory receptors for self-MHC is developmentally immature.

How receptor acquisition correlates with the functional maturation of natural killer (NK) cells is poorly understood. We used quantitative real-time polymerase chain reaction (PCR) assays to compare NKG2 and killer immunoglobulin-like receptor (KIR) gene expression in NK cells from allogeneic transplant recipients and their donors. Marked differences were observed in the NK subsets of recipients who had 8-fold more CD56(bright) cells, diminished KIR expression (except 2DL4), and increased NKG2A. In normal blood not all CD56(dim) cells express KIR, and a novel subpopulation of cells committed to the NK-cell lineage was defined. These cells, which comprise 19.4% +/- 2.8% of the CD56(dim) NK population in healthy donors, express the activating NKG2D and NKG2E receptors but no KIR or NKG2A. Although the CD56(dim) NKG2A(-) KIR(-) NK cells lack "at least one" inhibitory receptor for autologous MHC class I, they are not fully responsive, but rather functionally immature cells with poor cytotoxicity and IFN-gamma production. Upon culture with IL-15 and a stromal cell line, CD56(dim) and CD56(bright) KIR(-) NK cells proliferate, express KIR, and develop cytotoxicity and cytokine-producing potential. These findings have implications for the function of NK cells reconstituting after transplantation and support a model for in vivo development in which CD56(bright) cells precede CD56(dim) cells.

KIR reconstitution is altered by T cells in the graft and correlates with clinical outcomes after unrelated donor transplantation.

Although unrelated hematopoietic cell transplantation (HCT) is curative for many hematologic malignancies, complications and relapse remain challenging obstacles. Natural killer (NK) cells, which recover quickly after transplantation, produce cytokines and express killer immunoglobulin-like receptors (KIRs) that regulate their cytotoxicity. Some clinical trials based on a KIR ligand mismatch strategy are associated with less relapse and increased survival, but results are mixed. We hypothesized that T cells in the graft may affect NK cell function and KIR expression after unrelated transplantation and that these differences correlate with clinical outcomes. NK cell function was evaluated using 77 paired samples from the National Marrow Donor Program Research Repository. Recipient NK cells at 100 days after both unmanipulated bone marrow (UBM) and T-cell depleted (TCD) transplants were compared with NK cells from their healthy donors. NK cells expressed fewer KIRs and produced more interferon gamma (IFN-gamma) after UBM compared to TCD transplants. Multivariate models showed that increased NK cell IFN-gamma production correlated with more acute graft-versus-host disease (GVHD), and decreased KIR expression correlated with inferior survival. These results support the notion that T cells in the graft affect NK cell reconstitution in vivo. Understanding these mechanisms may result in strategies to improve clinical outcomes from unrelated HCT.