Cellular immunotherapy with multiple infusions of in vitro-expanded haploidentical natural killer cells after autologous transplantation for patients with plasma cell myeloma.

BACKGROUND AIMS: To investigate the feasibility and safety of haploidentical natural killer (NK) cell infusions as consolidation immunotherapy after autologous stem cell transplant (ASCT) in patients with plasma cell myeloma. METHODS: Ten patients (median age, 59 years) received induction treatment followed by high-dose melphalan (200 mg/m2) at day -1, ASCT at day 0 and increasing NK cell doses (1.5 × 106, 1.5 × 107 and multiple doses of 1.0 × 108 cells/kg body weight) from day +1 to day +30 after ASCT. NK cells were harvested and purified from peripheral blood of haploidentical donors and expanded for 19 days with interleukin (IL)-2 and IL-15 under Good Manufacturing Practice conditions. RESULTS: NK cell numbers increased 56.0-fold (37.4- to 75.5-fold). Patients received a median of 3.8 × 108 (0.9-5.7 × 108) NK cells/kg body weight in six (three to eight) infusions. Multiparametric mass cytometry analysis demonstrated an altered surface receptor repertoire of expanded NK cells with increased degranulation and cytokine production activities but diminished expression of perforin. Donor NK cells were detectable in the peripheral blood, peaking 1 h after each dose (up to 90% donor NK cells). The treatment was safe and well tolerated, without evidence of graft-versus-host disease. Comparison with a control patient population receiving ASCT without NK cell infusions showed no significant difference in relapse, progression-free survival and overall survival. CONCLUSIONS: This study demonstrates reliable manufacturing of high numbers of activated NK cells for multiple-dose infusions and safe administration of these cellular products. The trial was registered at ClinicalTrials.gov (identifier no. NCT01040026).

Good manufacturing practice-compliant cell sorting and large-scale expansion of single KIR-positive alloreactive human natural killer cells for multiple infusions to leukemia patients.

BACKGROUND AIMS: Alloreactive natural killer (NK) cells are potent effectors of innate anti-tumor defense. The introduction of NK cell-based immunotherapy to current treatment options in acute myeloid leukemia (AML) requires NK cell products with high anti-leukemic efficacy optimized for clinical use. METHODS: We describe a good manufacturing practice (GMP)-compliant protocol of large-scale ex vivo expansion of alloreactive NK cells suitable for multiple donor lymphocyte infusions (NK-DLI) in AML. CliniMACS-purified NK cells were cultured in closed air-permeable culture bags with certified culture medium and components approved for human use [human serum, interleukin (IL)-2, IL-15 and anti-CD3 antibody] and with autologous irradiated feeder cells. RESULTS: NK cells (6.0 ± 1.2 x 10(8)) were purified from leukaphereses (8.1 ± 0.8 L) of six healthy donors and cultured under GMP conditions. NK cell numbers increased 117.0 ± 20.0-fold in 19 days. To reduce the culture volume associated with expansion of bulk NK cells and to expand selectively the alloreactive NK cell subsets, GMP-certified cell sorting was introduced to obtain cells with single killer immunoglobulin-like receptor (KIR) specificities. The subsequent GMP-compliant expansion of single KIR+ cells was 268.3 ± 66.8-fold, with a contaminating T-cell content of only 0.006 ± 0.002%. The single KIR-expressing NK cells were cytotoxic against HLA-mismatched primary AML blasts in vitro and effectively reduced tumor cell load in vivo in NOD/SCID mice transplanted with human AML. CONCLUSIONS: The approach to generating large numbers of GMP-grade alloreactive NK cells described here provides the basis for clinical efficacy trials of NK-DLI to complement and advance therapeutic strategies against human AML.

Human acute myeloid leukemia CD34+CD38- stem cells are susceptible to allorecognition and lysis by single KIR-expressing natural killer cells.

The concept of tumor immunosurveillance has raised prospects for natural killer cell-based immunotherapy of human cancer. The cure of acute myeloid leukemia may depend on eradication of leukemic stem cells, the self-renewing component of leukemia. Whether natural killer cells can recognize and lyse leukemic stem cells is not known. To develop strategies that effectively target acute myeloid leukemia-leukemic stem cells, we investigated anti-leukemic effects of human alloreactive single KIR(+) natural killer cells. Natural killer effectors with KIR specificity mismatched with respect to HLA class I allotype of target cells effectively recognized acute myeloid leukemia-leukemic stem cells defined phenotypically as CD34(+)CD38(-), while healthy bone marrow-derived CD34(+)CD38(-) hematopoietic stem cells were spared, as demonstrated by cytotoxicity and hematopoietic colony-forming assays. The HDAC inhibitor valproic acid increased the activating NKG2D ligand-dependent lysis of acute myeloid leukemia-CD34(+)CD38(-) leukemic stem cells. These results show that alloreactive natural killer cells have the potential to detect and target leukemic stem cells, and thus to improve the treatment outcome in acute myeloid leukemia.

Differentiation-promoting drugs up-regulate NKG2D ligand expression and enhance the susceptibility of acute myeloid leukemia cells to natural killer cell-mediated lysis.

Natural killer (NK) cells are potent effectors of innate antitumor defense and are currently exploited for immune-based therapy of human leukemia. However, malignant blood cells in acute myeloid leukemia (AML) display low levels of ligands for the activating immunoreceptor NKG2D and can thus evade NK immunosurveillance. We examined the possibility of up-regulating NKG2D-specific UL16-binding protein (ULBP) ligands using anti-neoplastic compounds with myeloid differentiation potential. Combinations of 5-aza-2'-deoxycytidine, trichostatin A, vitamin D3, bryostatin-1, and all-trans-retinoic acid, used together with myeloid growth factors and interferon-gamma, increased cell surface ULBP expression up to 10-fold in the AML cell line HL60 and in primary AML blasts. Up-regulation of ULBP ligands was associated with induction of myelomonocytic differentiation of AML cells. Higher ULBP expression increased NKG2D-dependent sensitivity of HL60 cells to NK-mediated killing. These findings identify NKG2D ligands as targets of leukemia differentiation therapy and suggest a clinical benefit in combining a pharmacological approach with NK cell-based immunotherapy in AML.

Function of natural killer cells in immune defence against human leukaemia.

Although progress has been made in the management of acute leukaemias, most patients who fail to respond to front-line therapies with cytostatic agents and stem cell transplantation, or who relapse after an initial response die from progressive disease. Novel treatment modalities exploiting donor-derived natural killer (NK) cells generate an alloreactive graft-versus-leukaemia response and eliminate the residual malignant clones in transplanted patients. NK cells are components of the innate immunity playing an important role in the surveillance of human tumours. Recognition of malignant cells depends on a dynamic balance between antagonistic functions of an array of NK activating and inhibitory receptors. The natural cytotoxicity receptors (NCRs) are NK cell-specific and together with the NKG2D receptor are responsible for NK cell activation and tumour cell killing. The killer immunoglobulin-like receptors (KIRs) recruit phosphatases and can antagonise the activating signals and prevent the cytolytic NK cell programme. Understanding of the integration of these multiple signals at the molecular level is central for exploring the cytolytic function of NK cells. This review describes molecular mechanisms of NK receptor-ligand interactions controlling target cell recognition and addresses the potential of NK cells for the specific elimination of leukaemic clones with the goal of advancing immunotherapy of leukaemia.

The effect of silencing NKG2D through RNA interference on receptor functions in interleukin-2-activated human natural killer cells.

Natural killer (NK) cells are effectors of the innate immunity involved in tumor surveillance. NKG2D is a potent activating receptor eliciting cytokine and cytolytic NK responses upon recognition of tumor-associated ligands. We engineered primary interleukin (IL)-2-activated human NK cells to express constitutively low levels of NKG2D by lentiviral delivery of small interfering RNA. NKG2D-mediated effector functions were strongly impaired in NKG2D(low) NK cells. Reduction of NKG2D surface expression to 15%, corresponding to receptor levels in resting NK cells, rendered cells fully insensitive to NKG2D triggering. These data underscore the importance of NKG2D receptor cell surface density and suggest a threshold of expression for optimal reactivity of human NK cells.

Function of natural killer cells in immune defence against human leukaemia.

Although progress has been made in the management of acute leukaemias, most patients who fail to respond to front-line therapies with cytostatic agents and stem cell transplantation, or who relapse after an initial response die from progressive disease. Novel treatment modalities exploiting donor-derived natural killer (NK) cells generate an alloreactive graft-versus-leukaemia response and eliminate the residual malignant clones in transplanted patients. NK cells are components of the innate immunity playing an important role in the surveillance of human tumours. Recognition of malignant cells depends on a dynamic balance between antagonistic functions of an array of NK activating and inhibitory receptors. The natural cytotoxicity receptors (NCRs) are NK cell-specific and together with the NKG2D receptor are responsible for NK cell activation and tumour cell killing. The killer immunoglobulin-like receptors (KIRs) recruit phosphatases and can antagonise the activating signals and prevent the cytolytic NK cell programme. Understanding of the integration of these multiple signals at the molecular level is central for exploring the cytolytic function of NK cells. This review describes molecular mechanisms of NK receptor-ligand interactions controlling target cell recognition and addresses the potential of NK cells for the specific elimination of leukaemic clones with the goal of advancing immunotherapy of leukaemia.

Genetic modification of murine hematopoietic stem cells by retroviruses.

Among the currently available methods for gene transfer, recombinant murine retroviruses remain the best established method for achieving stable integration of a transgene with high efficiency. Pioneering work by a number of groups has demonstrated the feasibility of using this method for gene transfer to primitive, multipotential long-term repopulating hematopoietic stem cells (HSC) (1-4). In the case of the hematopoietic system, it is required that the introduced gene integrates into the genome of HSC in order to be expressed in multiple lineages over an extended period of time. However, HSC are found at low frequency, and are normally in a quiescent or slow cycling state. Both factors represent challenges to successful retroviral gene transfer. The former places a premium on high titer, and the latter dictates methods to trigger HSC cycling during the infection, since stable integration of murine retroviruses requires cell division of the target cell and breakdown of the nuclear membrane (5,6). In general, titers greater than 1 × 10(5) U/mL allow some degree of gene transfer for HSC, but 1 × 10(6) or higher are a reasonable goal for achieving useful efficiencies of at least 20%. For activation of HSC, most protocols invoke a combination of in vivo and in vitro stimulation. The former is most easily and routinely achieved by administration of cytotoxic agents like 5-fluorouracil (5-FU) 4 d prior to bone-marrow harvest. This procedure removes a large proportion of actively cycling, more differentiated cells, thus achieving a.

Post-transcriptional regulation of ULBP1 ligand for the activating immunoreceptor NKG2D involves 3' untranslated region.

The stress-inducible ULBP1 cell surface ligand for the activating immunoreceptor NKG2D allows recognition and lysis of tumor cells by natural killer (NK) and T cells. Understanding of mechanisms regulating ULBP1 expression is limited, but it is important for exploiting NKG2D-dependent antitumor responses. We studied the role of 3' untranslated region (3' UTR) in post-transcriptional regulation of ULBP1 expression in Jurkat and HeLa cells. Analysis of 2.4 kb-long 3' UTR revealed the presence of four AU-rich elements (ARE) and more then 200 putative microRNA binding sites. Stable or transient delivery of luciferase reporter constructs containing ULBP1-3' UTR sequences resulted in a strong reduction of luciferase activity to 7-22% with the full-length 3' UTR or 19%-62% with its fragments, indicating a contribution of 3' UTR to regulation of ULBP1 gene. Mutations introduced to ARE motifs significantly diminished luciferase activity, suggesting mRNA stabilizing effect of ARE. Among ULBP1-specific candidate microRNAs, we found miR-140-5p/-409-3p/-433-3p/-650 expressed in HeLa and Jurkat cells, and the microRNA involvement was supported by luciferase reporter assays with constructs carrying seed sequence mutations. However, microRNA overexpression or partial silencing of the microRNA processing enzyme Drosha did not equivocally clarify the role of microRNAs in regulation of ULBP1. Altogether these results provide evidence for a novel 3' UTR-mediated mechanism of regulation of ULBP1 at the post-transcriptional level.

Clinical stem-cell sources contain CD8+CD3+ T-cell receptor-negative cells that facilitate bone marrow repopulation with hematopoietic stem cells.

Clinical observations in patients undergoing bone marrow transplantation implicate the involvement of CD8(+) cells in promoting the stem-cell engraftment process. These findings are supported by mouse transplant studies, which attributed the engraftment-facilitating function to subpopulations of murine CD8(+) cells, but the analogous cells in humans have not been identified. Here, we report that clinical stem-cell grafts contain a population of CD8alpha(+)CD3epsilon(+) T-cell receptor- negative cells with an engraftment facilitating function, named candidate facilitating cells (cFCs). Purified cFC augmented human hematopoiesis in NOD/SCID mice receiving suboptimal doses of human CD34(+) cells. In vitro, cFCs cocultured with CD34(+) cells increased hematopoietic colony formation, suggesting a direct effect on clonogenic precursors. These results provide evidence for the existence of rare human CD8(+)CD3(+)TCR(-) cells with engraftment facilitating properties, the adoptive transfer of which could improve the therapeutic outcome of stem-cell transplantation.

NKG2D ligand expression in AML increases in response to HDAC inhibitor valproic acid and contributes to allorecognition by NK-cell lines with single KIR-HLA class I specificities.

This study exploited alloreactivity of natural killer (NK) cells for augmenting the recognition of human acute myeloid leukemia (AML). To circumvent the inhibitory effect of killer immunoglobulin receptor (KIR) signaling, we generated NK-cell lines with single KIR specificities for major human leukocyte antigen (HLA) class I allotypes. We demonstrated efficient cytolysis of KIR-HLA class I-mismatched primary AML blasts even at low effector-to-target ratios. To define the impact of tumor-associated activating NKG2D-ligands (NKG2D-L), 66 AML patients at diagnosis were analyzed. NKG2D-L were selectively expressed on monoblastic cells in AML M4 and M5 yet absent or weakly expressed on myeloblastic cells in all AML subtypes. Paucity of cell-surface NKG2D-L was not the result of shedding because levels of soluble ULBP1 ligand measured in AML plasma were in the normal range. Notably, purified NKG2D-L(+) monoblastic cells were more susceptible to NK-mediated killing than NKG2D-L(-) myeloblastic cells. Accordingly, induction of cell-surface NKG2D-L by treatment with the histone deacetylase inhibitor, valproic acid, rendered cells more sensitive to NK cytolysis. These data suggest that adoptive transfer of selected populations of alloreactive HLA class I-mismatched NK cells in combination with pharmacologic induction of NKG2D-L merits clinical evaluation as novel approaches to immunotherapy of human AML.

Normal erythropoiesis but severe polyposis and bleeding anemia in Smad4-deficient mice.

The tumor suppressor Smad4 mediates signaling by the transforming growth factor beta (TGF-beta) superfamily of ligands. Previous studies showed that several TGF-beta family members exert important functions in hematopoiesis. Here, we studied the role of Smad4 in adult murine hematopoiesis using the inducible Mx-Cre/loxP system. Mice with homozygous Smad4 deletion (Smad4(Delta/Delta)) developed severe anemia 6 to 8 weeks after induction (mean hemoglobin level 70 g/L). The anemia was not transplantable, as wild-type mice reconstituted with Smad4(Delta/Delta) bone marrow cells had normal peripheral blood counts. These mice did not develop an inflammatory disease typical for mice deficient in TGF-beta receptors I and II, suggesting that the suppression of inflammation by TGF-beta is Smad4 independent. The same results were obtained when Smad4 alleles were deleted selectively in hematopoietic cells using the VavCre transgenic mice. In contrast, lethally irradiated Smad4(Delta/Delta) mice that received wild-type bone marrow cells developed anemia similar to Smad4(Delta/Delta) mice that did not receive a transplant. Liver iron stores were decreased and blood was present in stool, indicating that the anemia was due to blood loss. Multiple polyps in stomach and colon represent a likely source of the bleeding. We conclude that Smad4 is not required for adult erythropoiesis and that anemia is solely the consequence of blood loss.

Ligands for natural killer cell-activating receptors are expressed upon the maturation of normal myelomonocytic cells but at low levels in acute myeloid leukemias.

Natural killer (NK) cell-mediated cytolytic activity against tumors requires the engagement of activating NK receptors by the tumor-associated ligands. Here, we have studied the role of NKG2D and natural cytotoxicity receptors (NCRs) in the recognition of human leukemia. To detect as-yet-unknown cell-surface molecules recognized by NCRs, we developed soluble forms of NKp30, NKp44, and NKp46 as staining reagents binding the putative cognate ligands. Analysis of UL16-binding protein-1 (ULBP1), ULBP2, and ULBP3 ligands for NKG2D and of potential ligands for NKp30, NKp44, and NKp46 in healthy hematopoietic cells demonstrated the ligand-negative phenotype of bone marrow-derived CD34(+) progenitor cells and the acquisition of cell-surface ligands during the course of myeloid differentiation. In acute myeloid leukemia (AML), leukemic blasts from approximately 80% of patients expressed very low levels of ULBPs and NCR-specific ligands. Treatment with differentiation-promoting myeloid growth factors, together with interferon-gamma, upregulated cell-surface levels of ULBP1 and putative NCR ligands on AML blasts, conferring an increased sensitivity to NK cell-mediated lysis. We conclude that the ligand-negative/low phenotype in AML is a consequence of cell maturation arrest on malignant transformation and that defective expression of ligands for the activating NKG2D and NCR receptors may compromise leukemia recognition by NK cells.

Human NK cell development in NOD/SCID mice receiving grafts of cord blood CD34+ cells.

Definition of the cytokine environment, which regulates the maturation of human natural killer (NK) cells, has been largely based on in vitro assays because of the lack of suitable animal models. Here we describe conditions leading to the development of human NK cells in NOD/SCID mice receiving grafts of hematopoietic CD34+ precursor cells from cord blood. After 1-week-long in vivo treatment with various combinations of interleukin (IL)-15, flt3 ligand, stem cell factor, IL-2, IL-12, and megakaryocyte growth and differentiation factor, CD56+CD3- cells were detected in bone marrow (BM), spleen, and peripheral blood (PB), comprising 5% to 15% of human CD45+ cells. Human NK cells of NOD/SCID mouse origin closely resembled NK cells from human PB with respect to phenotypic characteristics, interferon (IFN)-gamma production, and cytotoxicity against HLA class 1-deficient K562 targets in vitro and antitumor activity against K562 erythroleukemia in vivo. In the absence of growth factor treatment, CD56+ cells were present only at background levels, but CD34+CD7+ and CD34-CD7+ lymphoid precursors with NK cell differentiation potential were detected in BM and spleen of chimeric NOD/SCID mice for up to 5 months after transplantation. Our results demonstrate that limitations in human NK cell development in the murine microenvironment can be overcome by treatment with NK cell growth-promoting human cytokines, resulting in the maturation of IFN-gamma-producing cytotoxic NK cells. These studies establish conditions to explore human NK cell development and function in vivo in the NOD/SCID mouse model.

Gene silencing by lentivirus-mediated delivery of siRNA in human CD34+ cells.

To derive an efficient system for gene silencing in human hematopoietic stem cells (HSCs) we modified a lentiviral vector for small interfering RNA (siRNA) delivery. For this purpose, an H1 promoter-driven siRNA expression cassette was introduced into a lentiviral vector, and the p53 mRNA was chosen as a target for siRNA-mediated gene silencing. Using the recombinant lentivirus we infected human cord blood-derived CD34+ cells and obtained a transfection efficiency of up to 50%, as determined by expression of enhanced green fluorescent protein (EGFP). In EGFP-positive long-term culture-initiating cell (LTC-IC)- and colony-forming unit cell (CFU-C)-derived cells, we observed a reduction of p53 mRNA of up to 95%. Importantly, this reduction remained stable during several weeks of cell culture. Furthermore, p53 gene silencing resulted in decreased p21 mRNA levels and reduced the sensitivity of CD34+ cells toward the cytotoxic drug etoposide. Thus, lentiviral delivery of siRNA can allow for efficient and stable gene silencing in human HSCs and will be very valuable for further gene function studies.