ALBERT

Description: 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 CD56bright cells, diminished KIR expression (except 2DL4), and increased NKG2A. In normal blood not all CD56dim 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 CD56dim NK population in healthy donors, express the activating NKG2D and NKG2E receptors but no KIR or NKG2A. Although the CD56dim 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-γ production. Upon culture with IL-15 and a stromal cell line, CD56dim and CD56bright 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 CD56bright cells precede CD56dim cells.

Description: The stage of progenitor maturation and factors that determine the fate and clonal acquisition of human natural killer (NK) cell receptors during development are unknown. To study human NK cell receptor ontogeny, umbilical cord blood CD34+/Lin−/CD38− cells were cultured with a murine fetal liver line (AFT024) and defined cytokines. In the absence of lymphocyte-stimulating cytokines or when contact with AFT024 was prohibited, NK cell progeny were killer immunoglobulinlike receptor (KIR) and CD94 lectin receptor negative. In contrast, efficient NK cell differentiation and receptor acquisition was dependent on direct contact of progenitors with AFT024 and the addition of interleukin-15 (IL-15) or IL-2 but not IL-7. To address the question of whether receptor acquisition was determined at the stem cell level, single CD34+/Lin−/CD38−progenitors were studied. More than 400 single cell progeny were analyzed from cultures containing IL-15 or IL-2 and NK cells were always polyclonal, suggesting that receptor fate is determined beyond an uncommitted progenitor and that receptor-negative NK cells acquire class I-recognizing receptors after lineage commitment. KIR2DL2/L3/S2 was expressed more than KIR2DL1/S1 or KIR3DL1, and NKG2A was the dominant CD94 receptor, independent of whether the stem cell source contained the respective major histocompatibility complex class I ligand, suggesting a nonrandom sequence of receptor acquisition. The conclusion is that NK receptor fate is determined after NK cell commitment, does not require stromal presentation of human class I alleles, and is clonally stable after expression but dynamic because new receptors are acquired over time.

Description: We have demonstrated that long-term culture initiating cells (LTC-IC) are maintained in a stroma noncontact (SNC) culture where progenitors are separated from stroma by a microporous membrane and LTC-IC can proliferate if the culture is supplemented with interleukin-3 (IL-3) and macrophage inflammatory protein-1α (MIP-1α). We hypothesize that the same conditions, which result in LTC-IC proliferation, may also maintain lymphoid progenitors. Natural killer (NK) cells are of lymphoid lineage and a stromal-based culture can induce CD34+/Lin−/DR−cells to differentiate along the NK cell lineage. We developed a three-step switch culture assay that was required to demonstrate the persistence of NK progenitors in CD34+/Lin−/DR− cells assayed in SNC cultures supplemented with IL-3 and MIP-1α. When CD34+/Lin−/DR− progeny from the SNC culture were plated sequentially into “NK cell progenitor switch” conditions (contact with stromal ligands, hydrocortisone-containing long-term culture medium, IL-2, IL-7, and stem cell factor [SCF]) followed by “NK cell differentiation” conditions (contact with stromal ligands, human serum, no hydrocortisone, and IL-2), significant numbers of CD56+/CD3− NK resulted, which exhibited cytotoxic activity against K562 targets. All steps are required because a switch from SNC cultures with IL-3 and MIP-1α directly to “NK cell differentiation” conditions failed to yield NK cells suggesting that critical step(s) in lymphoid commitment were missing. Additional experiments showed that CD34+/CD33− cells present after SNC cultures with IL-3 and MIP-1α, which contained up to 30% LTC-IC, are capable of NK outgrowth using the three-step switch culture. Limiting dilution analysis from these experiments showed a cloning frequency within the cultured CD34+/CD33− population similar to fresh sorted CD34+/Lin−/DR− cells. However, after addition of FLT-3 ligand, the frequency of primitive progenitors able to develop along the NK lineage increased 10-fold. In conclusion, culture of primitive adult marrow progenitors ex vivo in stroma-derived soluble factors, MIP-1α, and IL-3 maintains both very primitive myeloid (LTC-IC) and lymphoid (NK) progenitors and suggests that these conditions may support expansion of human hematopoietic stem cells. Addition of FLT-3 ligand to IL-2, IL-7 SCF, and stromal factors are important in early stages of NK development.

Description: We have previously shown that adoptive transfer of haploidentical natural killer (NK) cells can induce remissions in 27% of patients with refractory or relapsed acute myeloid leukemia (AML) [Miller et al., Blood 2005, 105 (8)]. Aiming to optimize NK cell expansion, which we hypothesize is required for therapeutic efficacy, we tested additional CD56-positive selection (N=10) versus the CD3-depletion method used for our earlier NK cell infusions (N=10). Donor-derived NK cells were not measurable immediately after infusion. Successful in vivo NK cell expansion, defined as 〉100 donor-derived NK cells/ml at 14 days (by VNTR chimerism and flow cytometry) was not improved with CD56-selection (11% vs. 11%; mean 131±3 NK cells/ml), and was associated with poorer outcomes (10% vs. 27% AML remissions). Because the remissions induced by adoptive NK cell transfer were not durable, we added a CD34+ stem cell infusion to create a nonmyeloablative haploidentical transplantation protocol for older and less fit patients. We also added radiation to the NK cell-based preparative regimen to further improve NK cell expansion. The lymphodepleting chemoradiation plus NK cell preparative regimen included fludarabine 25 mg/m2 × 5 (day -18 through day -14), cyclophosphamide 60 mg/kg × 2 (days -16 and -15), and 200 cGy of total body irradiation (twice a day on day -13). The NK cell product, prepared by cliniMACS (Miltenyi) CD3-depletion of a single leukapheresis collection from a haploidentical related donor, was incubated overnight in 1000 U/ml IL-2 and then infused on day -12 followed by 6 doses subcutaneous IL-2 (10 million units) given every other day to promote in vivo NK cell expansion. The mean NK cell dose was 1.85 × 107 cells/kg and the mean CD3+ cell dose was 4.8 × 104 cells/kg (maximum permitted 3 × 105 CD3+ cells/kg). A CD34-selected filgrastim-mobilized peripheral blood graft from the same donor (target dose 〉3 × 106 CD34 cells/kg) was given with Thymoglobulin 3 mg/kg days 0, +1 and +2 as the only additional immunosuppression. In the 13 patients treated to date a significantly higher rate of NK cell expansion (75% [9/12 evaluable]; mean 607±184 NK cells/ml) was achieved compared to the adoptive NK cell transfer regimen, which did not include radiation. Plasma IL-15, which is critical for NK expansion, was highest on day -12 (the NK infusion day) after the preparative regimen (64 ± 8 pg/ml [day -12] vs. 6 ± 1 pg/ml [baseline pre-chemo]; p 500/ml and 100% donor chimerism: median 17 days [range 11–31]). None developed graft vs. host disease (GVHD), but infections were common (3 fatal EBV/PTLD; 1 Fusarium). To prevent EBV reactivation NK products are now CD19 depleted and patients receive prophylactic Rituxan to prevent PTLD. The other deaths were due to persistent disease (N=4) or relapse (N=1). One patient is alive in remission beyond day +100. No clear associations between killer immunoglobulinlike receptor (KIR) ligand mismatch between donor and recipient were detected. In this series of patients with refractory AML, addition of haploidentical NK cells to a nonmyeloablative haploidentical transplantation yields NK cell expansion in a majority of patients, achievement of complete remission, and quick engraftment without GVHD. This is a promising platform upon which to add other strategies aimed at improving disease free survival in patients with refractory AML.

Description: Abstract 346 Expansion of NK cells after adoptive transfer is a major determinant of their anti-tumor efficacy but the mechanisms of their expansion are not completely understood. To study in vivo NK cell expansion and the relative merits of fresh versus frozen NK cell products, we used xenotransplantation of human GMP NK cell products (provided by two PACT centers funded by the NHLBI) into immune deficient mice. NOD/IL-2Rγc/Rag (NOG) that lack T-, B-, and NK cells and that have a macrophage defect that renders recipients highly amenable to human cell engraftment, were given 250 cGy total body irradiation, and infused with fresh or frozen NK cells. Two products were tested: 1) an enriched fresh NK cell product obtained by CD3 and CD19 depletion followed by overnight activation with IL-2 or IL-15 (U. of Minnesota) or 2) ex vivo expanded NK cells using membrane bound IL-15 and 41BB-L transduced K562 cells with 10 U/ml IL-2 in G-Rex devices for 10 days (Baylor). NK cells (1–2 million NK cells for each individual experiment) were given IV and mice were treated with no cytokines, 5 or 10 mg of IL-2 (Novartis), or 5 mg IL-15 (produced by the NCI for clinical use) as 3 injections per week for 2 weeks. Peripheral blood (Table), spleen and marrow were evaluated on days 7, 14, 21 and 28 after adoptive transfer. Counts were converted to absolute cell numbers per 100 μL of blood or the number of cells recovered per spleen or after flushing of a single femur. On day 7, the number of huNK cells in blood in the absence of cytokine administration was low with both cell products and numbers continued to diminish by day 14. In marked contrast, every other day IL-2 or IL-15 significantly increased huNK cells in murine blood. On day 7 and 14, ex vivo expanded NK cell products resulted in significantly higher numbers of huNK in mice receiving 5μg IL-2 compared to mice who received fresh NK cells activated overnight with IL-2. With 5μg IL-2, huNK number decreased between day 7 and 14 irrespective of cell product. In contrast, only IL-15 lead to increased numbers of huNK cells between days 7 and 14. Previously cryopreserved NK cells showed significantly worse survival for both cell products, with a larger fold decrease seen with ex vivo expanded cells. Blood collected from cytokine treated mice at day 14 after infusion of fresh products contained huNK cells that were fully functional as assessed by potent CD107a degranulation, TNF and IFN production after exposure to K562 target cells as well as augmented IFN production induced by IL-12 and IL-18. On day 8, Ki67+ (proliferating) huNK cells were significantly higher with IL-15 compared to IL-2 (both at 5μg) in marrow for fresh (76% vs 35%, p=

Description: Effective therapeutic options are lacking for patients with primary refractory or relapsed acute myelogenous leukemia (AML). We have shown that adoptive transfer of haploidentical NK cells can result in complete remissions (25%) in patients with refractory disease but this procedure is still limited by a high failure rate. Based on the finding that 29±3% (n=26) of normal CD56dim NK cells are KIR− NKG2A+, we hypothesized that non-KIR class I MHC inhibitory receptors may play a bigger role than previously recognized in the cumulative integration of signals that determine whether leukemia targets are killed by NK cells. We studied the role of NKG2A and KIR inhibitory signals on primary AML and acute lymphoblastic leukemia (ALL) targets collected by therapeutic lymphapheresis. Blast susceptibility to fresh resting polyclonal allogeneic NK cells (enriched by CD3 depletion) and to NK cells activated with low pharmacologic doses of IL-2 for 72 hours was determined using flow cytometry based cytotoxicity assays and a degranulation assay using CD107a. The role of inhibitory signaling through class I MHC-recognizing receptors was tested by blocking interactions between receptor and cognate ligand using 1) a pan-MHC monoclonal antibody (mAb) (clone HP17-F) recognizing HLA-A, B, C and E, 2) an anti-KIR reagent (1-7F9 which blocks inhibitory KIR2DL1/L2/L3) currently in clinical trials (Novonordisk, Copenhagen) and 3) a mAb against NKG2A (clone Z199). Susceptibility to killing was defined as 〉10% lysis at an E:T ratio of 10:1. Two of 8 leukemias (25%) were lysed by resting allogeneic NK cells. NK cells activated with IL-2 (5U/ml) killed 7 of 8 targets (average increase of 13±2.2% lysis). In 3 AMLs killing was significantly enhanced by pan-MHC mAb blockade, but was less enhanced by anti-KIR blockade. NKG2A blockade alone also increased killing, and when added to anti-KIR blockade AML killing was equal to that obtained with MHC blockade. This suggests that non-KIR class I recognizing interactions were operant. Interestingly, anti-KIR blockade did significantly enhance killing of ALL targets suggesting that higher MHC expression (MFI 3-fold greater) may be a dominant factor to NK cell susceptibility in ALL. These findings support the notion that susceptibility to NK cell lysis is determined by interactions beyond just KIR and KIR-ligands. To explore this further KIR− NK cells were enriched with immunomagnetic bead separation (80% NKG2A+). AML target killing by these IL-2 activated KIR−NKG2A+ NK cells increased significantly after pan-MHC and NKG2A blockade (from an average baseline of 21% lysis to 34% for both), but no increase was seen with KIR blockade. This suggests that a significant proportion of normal NK cells are negatively regulated by KIR-independent mechanisms interacting with HLA-E. In summary, interrupting dominant NK cell receptor interactions with class I MHC ligands may lead to better strategies to treat AML and even ALL. NKG2A blockade may be a good target as it is highly expressed on virtually all NK cells reconstituting in the first 3 months after allogeneic HCT and it identifies NK cells with effector function in normal subjects (Cooley et al, Blood110:5782007). The combination of inhibitory signal blockade with other activators such as low dose IL-2 or mAb capable of ADCC may be needed to best exploit the clinical therapeutic potential of NK cells.

Description: Marrow stromal cultures support adult CD34+/Lin−/HLA-DR− or CD34+/Lin−/CD38− cell differentiation into natural killer (NK) or myeloid cells, but unlike committed lymphoid progenitors (CD34+/Lin−/CD45RA+/CD10+), no B cells are generated. We tested whether different microenvironments could establish a developmental link between the NK and B-cell lineages. Progenitors were cultured in limiting dilutions with interleukin-7 (IL-7), flt3 ligand (FL), c-kit ligand (KL), IL-3, IL-2, and AFT024, a murine fetal liver line, which supports culture of transplantable murine stem cells. NK cells, CD10+/CD19+ B-lineage cells and dendritic cells (DC) developed from the same starting population and IL-7, FL, and KL were required in this process. Single cell deposition of 3,872 CD34+/Lin−/CD38− cells onto AFT024 with IL-7, FL, KL, IL-2, and IL-3 showed that a one time addition of IL-3 at culture initiation was essential for multilineage differentiation from single cells. Single and double lineage progeny were frequently detected, but more importantly, 2% of single cells could give rise to at least three lineages (NK cells, B-lineage cells, and DC or myeloid cells) providing direct evidence that NK and B-lineage differentiation derive from a common lymphomyeloid hematopoietic progenitor under the same conditions. This study provides new insights into the role of the microenvironment niche, which governs the earliest events in lymphoid development.

Description: Abstract 3611 Adoptive transfer of haploidentical natural killer (NK) cells can induce remissions in patients with refractory myeloid leukemia (AML). However, NK cells do not expand and persist in all patients despite lymphodepleting chemotherapy. In trials of adoptive NK cell therapy in solid tumors or lymphoma, host regulatory T cells (Treg) often expand in response to IL-2 given to stimulate donor NK cell expansion. Although murine studies report that Tregs inhibit NK cells, the influence of human Treg on NK cell proliferation and function is not well characterized. We studied the effect of allogeneic Tregs that were derived from human umbilical cord blood (UCB) as described by our group. Resting CFSE labelled NK cells or Teff were purified from healthy donors, and mixed with UCB Treg at various ratios. Unstimulated NK cells did not proliferate and thus IL-2 or IL-15 were added to the media at concentrations of 0.1, 0.25 and 0.5 ng/ml. In the absence of Treg, both cytokines induced equal NK cell proliferation at 5 days as measured by CFSE dilution in a concentration dependent manner. CFSE dilution was inhibited by Treg at a 1:1 ratio, especially at low cytokine concentrations. There were marked differences between the two cytokine conditions. Following IL-15 induced stimulation, the reduction in NK cell proliferation by Treg ranged from 1–35% (at different concentrations tested), whereas the inhibition of IL-2 stimulated NK cell proliferation ranged from 65–85%. Treg inhibition of NK cell proliferation could be measured at ratios as low as 1:8 in the presence of IL-2, but not IL-15. This inhibitory effect was partially explained by competition from CD25+ Tregs for IL-2. We measured Treg utilization of IL-2 by incubating NK cells with or without Treg in 0.5 ng/ml IL-2 for 4 days. The level of IL-2 with NK cells alone was 40 pg/ml vs. 17 pg/ml with Treg (compared to 330 pg/ml in IL-2-supplemented media without cells). Based on this data, we have incorporated host Treg depletion to enhance NK expansion after adoptive transfer to treat patients with refractory AML. As murine data from Blazar's group shows that CTL therapy is enhanced by Treg depletion, we added one dose of denileukin diftitox (ONTAK®, Eisai Inc) at 12 mg/kg to our lymphodepleting preparative regimen of fludarabine 25 mg/m2 × 5 days, cyclophosphamide 60 mg/kg × 2 days for 12 AML patients. Haploidentical NK cells (CD3- and CD19-depleted PBMCs and overnight activated with IL-2 1000 U/ml) were infused on Day 0, followed by 6 doses subcutaneous IL-2 (9 million units) given every other day to promote in vivo NK cell expansion. Eleven of 12 patients were evaluable, having received at least 4 of 6 planned doses of IL-2. Blood and marrow were collected 7 and 14 days after infusion to assess NK cell and Treg expansion, as well as leukemia clearance. Of the 10 patients with interpretable day 7 chimerism data, 9 had detectable donor DNA (median 68% donor DNA). At day 14, 4 of the 12 patients (33%) had successfully expanded NK cells in vivo, with absolute donor derived NK cell counts of 480, 530, 1470 and 12390 cells/μL blood, improving on our previous 10% rate of in vivo NK cell expansion which was observed with the same regimen, without Treg depletion. In the 4 patients who expanded NK cells in vivo, there were no detectable Treg (defined as a CD25+CD4+FoxP3+ lymphocyte population) at either day 7 or day 14. In contrast, the presence of a bona fide Treg population at either day 7 [range 9.5–53%] or day 14 [27–71%] correlated with a lack of in vivo NK cell expansion at day 14. Clinically, 8 of the 11 evaluable subjects cleared leukemia (72%), 7 of whom recovered neutrophils (63% CRp) and 6 of whom went on to best donor transplant (45%). In summary, we demonstrate in vitro and in vivo suppression of NK cell proliferation by IL-2 stimulated Treg. This effect is not seen in vitro with IL-15. We have shown that the absence of host Treg correlates with in vivo NK cells expansion. Although an increased rate of donor NK expansion was observed with a single dose of denileukin diftitox, it did not completely overcome the IL-2 induced host Treg expansion. Future trials testing additional doses of denileukin difitox or other methods of Treg depletion, as well as the use of IL-15 are planned. Disclosures: No relevant conflicts of interest to declare.

Description: Following allogeneic hematopoietic cell transplantation, NK cells play important roles in hematopoietic cell engraftment, anti-viral responses, graft vs. host disease (GVHD) and graft vs. leukemia (GVL) reactions. Calcineurin inhibitors, such as cyclosporine A (CsA), are frequently administered to prevent or treat GVHD. It is generally considered that these immune suppressive agents inhibit GVL reactions. To date, little is known about the impact of these immune suppressants on NK cell function. To investigate this, NK cells were isolated from normal donors by negative selection and cultured with IL-2 (100 U/ml), IL-15 (10 ng/ml) and either physiological levels of CsA (1μg/ml) or vehicle control. Under these conditions, we consistently found that CsA treated cultures showed a reduction in NK cell expansion at one week (4.88 vs. 1.87 fold expansion, n=10). The phenotype of CsA treated NK cells was markedly different than controls. More specifically, after 7–10 days of culture with CsA there were significantly more CD56brightCD16− cells and significantly less CD56dimCD16+ cells compared to controls (p

Description: We have demonstrated that long-term culture initiating cells (LTC-IC) are maintained in a stroma noncontact (SNC) culture where progenitors are separated from stroma by a microporous membrane and LTC-IC can proliferate if the culture is supplemented with interleukin-3 (IL-3) and macrophage inflammatory protein-1α (MIP-1α). We hypothesize that the same conditions, which result in LTC-IC proliferation, may also maintain lymphoid progenitors. Natural killer (NK) cells are of lymphoid lineage and a stromal-based culture can induce CD34+/Lin−/DR−cells to differentiate along the NK cell lineage. We developed a three-step switch culture assay that was required to demonstrate the persistence of NK progenitors in CD34+/Lin−/DR− cells assayed in SNC cultures supplemented with IL-3 and MIP-1α. When CD34+/Lin−/DR− progeny from the SNC culture were plated sequentially into “NK cell progenitor switch” conditions (contact with stromal ligands, hydrocortisone-containing long-term culture medium, IL-2, IL-7, and stem cell factor [SCF]) followed by “NK cell differentiation” conditions (contact with stromal ligands, human serum, no hydrocortisone, and IL-2), significant numbers of CD56+/CD3− NK resulted, which exhibited cytotoxic activity against K562 targets. All steps are required because a switch from SNC cultures with IL-3 and MIP-1α directly to “NK cell differentiation” conditions failed to yield NK cells suggesting that critical step(s) in lymphoid commitment were missing. Additional experiments showed that CD34+/CD33− cells present after SNC cultures with IL-3 and MIP-1α, which contained up to 30% LTC-IC, are capable of NK outgrowth using the three-step switch culture. Limiting dilution analysis from these experiments showed a cloning frequency within the cultured CD34+/CD33− population similar to fresh sorted CD34+/Lin−/DR− cells. However, after addition of FLT-3 ligand, the frequency of primitive progenitors able to develop along the NK lineage increased 10-fold. In conclusion, culture of primitive adult marrow progenitors ex vivo in stroma-derived soluble factors, MIP-1α, and IL-3 maintains both very primitive myeloid (LTC-IC) and lymphoid (NK) progenitors and suggests that these conditions may support expansion of human hematopoietic stem cells. Addition of FLT-3 ligand to IL-2, IL-7 SCF, and stromal factors are important in early stages of NK development.