Andreas Lundqvist

Clinical-grade ex vivo-expanded human natural killer cells up-regulate activating receptors and death receptor ligands and have enhanced cytolytic activity against tumor cells

BACKGROUND AIMSnCancer immunotherapy involving natural killer (NK) cell infusions and administration of therapeutic agents modulating the susceptibility of tumors to NK-cell lysis has been proposed recently. We provide a method for expanding highly cytotoxic clinical-grade NK cells in vitro for adoptive transfer following bortezomib treatment in patients with advanced malignancies.nnnMETHODSnNK cells were expanded with irradiated Epstein-Barr virus-transformed lymphoblastoid cells. Expanded cells were evaluated for their phenotype, cytotoxicity, cytokine secretion, dependence on interleukin (IL)-2 and ability to retain function after cryopreservation.nnnRESULTSnA pure population of clinical-grade NK cells expanded 490+/-260-fold over 21 days. Expanded NK cells had increased TRAIL, FasL and NKG2D expression and significantly higher cytotoxicity against bortezomib-treated tumors compared with resting NK cells. Expanded NK cells, co-cultured with K562 and renal cell carcinoma tumor targets, secreted significantly higher levels of soluble Fas ligand 6; fgjhd IFN-gamma, GM-CSF, TNF-alpha, MIP-1alpha and MIP-1beta compared with resting NK cells. Secretion of the above cytokines and NK-cell cytolytic function were IL-2 dose dependent. Cryopreservation of expanded NK cells reduced expression of NKG2D and TRAIL and NK-cell cytotoxicity, although this effect could be reversed by exposure of NK cells to IL-2.nnnCONCLUSIONSnWe describe a method for large-scale expansion of NK cells with increased expression of activating receptors and death receptor ligands resulting in superior cytotoxicity against tumor cells. This ex vivo NK-cell expansion technique is currently being utilized in a clinical trial evaluating the anti-tumor activity of adoptively infused NK cells in combination with bortezomib.

Bortezomib treatment and regulatory T-cell depletion enhance the antitumor effects of adoptively infused NK cells

Ligation of inhibitory receptors renders natural killer (NK) cells inactive against autologous tumors. Recently, the proteasome inhibitor bortezomib was shown to sensitize tumors to autologous NK-cell cytotoxicity in vitro. Here, we show bortezomib augments the antitumor effects of syngeneic NK-cell infusions in tumor-bearing animals; this effect is further enhanced in regulatory T cell (Treg cell)-depleted hosts. In vitro, bortezomib-treated tumors had higher tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and perforin/granzyme-mediated caspase-8 activity, which enhanced their susceptibility to NK-cell lysis. Bioluminescence imaging of mice with established tumors showed treatment with bortezomib and syngeneic NK cells reduced tumor growth and prolonged survival compared with controls receiving bortezomib or NK cells alone. In contrast, tumor progression was not delayed when animals received bortezomib and perforin-deficient NK cells, showing drug-induced augmentation in NK-cell cytotoxicity was mediated through perforin/granzyme. Furthermore, tumor growth was slower in bortezomib-treated recipients when host Treg cells were eradicated with anti-CD25 antibody before infusing NK cells compared with mice without Treg-cell ablation (tumor doubling time, 16.7 vs 4.9 days, respectively; P = .02). These findings suggest that depletion of Treg cells followed by bortezomib-induced tumor sensitization to autologous NK cells could be used as a novel strategy to treat cancer.

Natural killer cell immunotherapy for cancer: a new hope

Recently there has been a substantial gain in our understanding of the role NK-cells play in mediating innate host immune responses. Although NK cells have long been known to mediate antigen independent tumor cytotoxicity, the therapeutic potential of NK cell-based immunotherapy has yet to be realized. Manipulating the balance between inhibitory and activating NK receptor signals, sensitization of tumor target cells to NK cell-mediated apoptosis, and recent discoveries in NK-cell receptor biology have fueled translational research that has led to clinical trials investigating a number of novel methods to potentiate NK cytotoxicity against human malignancies.

Fetal and adult multipotent mesenchymal stromal cells are killed by different pathways.

BACKGROUND AIMSnMultipotent mesenchymal stromal cells, also known as mesenchymal stem cells (MSC), can be isolated from adult and fetal tissues. Recently, there has been considerable interest in MSC because they have features favorable for transplantation, namely their multipotency and non-immunogenic properties.nnnMETHODSnWe analyzed how human MSC derived from first-trimester fetal liver and adult bone marrow interact with naive and activated innate natural killer (NK) cells. NK cell function was studied by measuring killing of MSC, as well as degranulation (CD107a) induced by MSC. To assess the importance of NK cell killing, expression of surface epitopes was analyzed by flow cytometry on MSC before and after stimulation with interferon (IFN)γ.nnnRESULTSnFetal and adult MSC express several ligands to activating NK cell receptors as well as low levels of HLA class I, with large inter-individual variation. Naive peripheral blood NK cells did not lyse fetal or adult MSC, whereas interleukin (IL)2 activated allogeneic as well as autologous NK cells did. Pre-incubation of MSC with IFN-γ increased their levels of HLA class I, protecting them from NK cell recognition. Fetal and adult MSC were preferably killed via the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL) pathways, respectively. Blocking NKG2D reduced NK cell degranulation in both fetal and adult MSC.nnnCONCLUSIONSnFetal and adult MSC differ in their interactions with NK cells. Both fetal and adult MSC are susceptible to lysis by activated NK cells, which may have implications for the use of MSC in cell therapy.

Primitive quiescent CD34+ cells in chronic myeloid leukemia are targeted by in vitro expanded natural killer cells, which are functionally enhanced by bortezomib.

Primitive quiescent CD34(+) chronic myeloid leukemia (CML) cells are more biologically resistant to tyrosine kinase inhibitors than their cycling counterparts; however, graft-versus-leukemia (GVL) effects after allogeneic stem cell transplantation (SCT) probably eliminate even these quiescent cells in long-term surviving CML transplant recipients. We studied the progeny of CD34(+) cells from CML patients before SCT, which were cultured 4 days in serum-free media with hematopoietic growth factors. BCR-ABL expression was similar in both cycling and quiescent noncycling CD34(+) populations. Quiescent CD34(+) cells from CML patients were less susceptible than their cycling CD34(+) and CD34(-) counterparts to lysis by natural killer (NK) cells from their HLA-identical sibling donors. Compared with cycling populations, quiescent CD34(+) CML cells had higher surface expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5. Bortezomib up-regulated TRAIL receptor expression on quiescent CD34(+) CML cells, and further enhanced their susceptibility to cytotoxicity by in vitro expanded donor NK cells. These results suggest that donor-derived NK cell-mediated GVL effects may be improved by sensitizing residual quiescent CML cells to NK-cell cytotoxicity after SCT. Such treatment, as an adjunct to donor lymphocyte infusions and pharmacologic therapy, may reduce the risk of relapse in CML patients who require treatment by SCT.

Cutting edge: bortezomib-treated tumors sensitized to NK cell apoptosis paradoxically acquire resistance to antigen-specific T cells.

Bortezomib augments caspase-8 activity, rendering tumors susceptible to NK cell lysis. We hypothesized this effect would likewise sensitize tumors to Ag-specific CTLs. Instead, bortezomib-treated tumors that acquired sensitivity to NK cells simultaneously became resistant to killing by Ag-specific CTLs. Reduction in CTL killing persisted for days, was not due to changes in tumor expression of MHC class I, and was overcome by pulsing tumors with peptides recognized by tumor-reactive CTLs. Tumor-outgrowth experiments showed tumors grew faster in SCID mice when cocultures of tumor-reactive CTLs and bortezomib-treated tumors were injected compared with untreated tumors (tumor doubling time 3.1 and 10.6 d, respectively; p < 0.01), whereas tumors grew slower in mice receiving cocultures of NK cells and bortezomib-treated tumors compared with untreated tumors (11.8 d and 5.0 d, respectively; p < 0.01). These findings demonstrate bortezomib-treated tumors sensitized to NK cell apoptosis paradoxically acquire resistance to CTLs as a consequence of bortezomib altering proteasomal processing and presentation of tumor Ags.

Differences in the Phenotype, Cytokine Gene Expression Profiles, and In Vivo Alloreactivity of T Cells Mobilized with Plerixafor Compared with G-CSF

Plerixafor (Mozobil) is a CXCR4 antagonist that rapidly mobilizes CD34+ cells into circulation. Recently, plerixafor has been used as a single agent to mobilize peripheral blood stem cells for allogeneic hematopoietic cell transplantation. Although G-CSF mobilization is known to alter the phenotype and cytokine polarization of transplanted T cells, the effects of plerixafor mobilization on T cells have not been well characterized. In this study, we show that alterations in the T cell phenotype and cytokine gene expression profiles characteristic of G-CSF mobilization do not occur after mobilization with plerixafor. Compared with nonmobilized T cells, plerixafor-mobilized T cells had similar phenotype, mixed lymphocyte reactivity, and Foxp3 gene expression levels in CD4+ T cells, and did not undergo a change in expression levels of 84 genes associated with Th1/Th2/Th3 pathways. In contrast with plerixafor, G-CSF mobilization decreased CD62L expression on both CD4 and CD8+ T cells and altered expression levels of 16 cytokine-associated genes in CD3+ T cells. To assess the clinical relevance of these findings, we explored a murine model of graft-versus-host disease in which transplant recipients received plerixafor or G-CSF mobilized allograft from MHC-matched, minor histocompatibility–mismatched donors; recipients of plerixafor mobilized peripheral blood stem cells had a significantly higher incidence of skin graft-versus-host disease compared with mice receiving G-CSF mobilized transplants (100 versus 50%, respectively, p = 0.02). These preclinical data show plerixafor, in contrast with G-CSF, does not alter the phenotype and cytokine polarization of T cells, which raises the possibility that T cell–mediated immune sequelae of allogeneic transplantation in humans may differ when donor allografts are mobilized with plerixafor compared with G-CSF.

Abstract 1271: In vitro expanded natural killer (NK) cells are more susceptible to Fas-mediated apoptosis compared to fresh and overnight IL-2 activated NK cells

Introduction: NK cells are currently being explored as a treatment modality for many oncologic diseases. However, the small number of NK cells in the circulation, approximately 5-15%, may limit the effectiveness of host NK cell immunity against cancer. Several methods to expand NK cells for subsequent adoptive infusion in cancer patients have been explored, including culturing NK cells in vitro with interleukin-2 (IL-2). Unfortunately, IL-2 stimulation alone results in only a small increase in NK cell proliferation. Recently, a novel in vitro method for large scale NK cell expansions using irradiated EBV-LCL feeder cells has been developed and is currently being utilized in an NHLBI clinical trial. Purpose: The aims of this study included: a) to identify differences in susceptibility to apoptotic pathways between fresh, overnight IL-2 activated, and expanded NK cells by comparing the death receptors DR4, DR5, and Fas; b) to determine when Fas expression is acquired on IL-2 activated NK cells; and c) to determine if Fas expression differs on NK cells expanded with different feeder cell populations. Methods: NK cells from healthy donors were isolated using CD56+ magnetic selection beads. Using flow cytometry, we examined Fas, DR4, and DR5 expression on fresh, overnight IL-2 activated, and NK cells expanded with either EBV-LCL or K562 41bb ligand transfected feeder cells for 7-14 days. To assess susceptibility to Fas mediated apoptosis, each NK cell group was cultured with recombinant FasL (rhFasL) for 12 hours with Annexin V expression by flow cytometry used to measure apoptosis. In addition, Fas expression was examined over multiple days for both IL-2 activated NK cells and expanded NK cells. Results: Fas expression was similar on overnight IL-2 activated NK cells compared to fresh NK cells. In contrast, NK cells maintained in IL-2 for more than 2 days and expanded NK cells had a substantial increase in Fas expression. Similar increases in Fas expression were seen in NK cells expanded with either EBV-LCL or K562 41bb ligand transfected feeder cells. Enhanced Fas expression was associated with increased susceptibility to rhFasL mediated apoptosis; the fold increase in Annexin V induced by rhFasL at 12 hours was significantly higher amongst expanded NK cells (5.6 fold increase; p = 0.0001) compared to either fresh NK cells (1.2 fold increase; p = 0.32) or overnight IL-2 activated NK cells (1.0 fold increase; p = 0.62). In contrast to Fas, surface expression of the TRAIL death receptors DR4 and DR5 were similar amongst all NK cell populations analyzed. Conclusion: These data show for the first time that expanded NK cells are more susceptible to Fas mediated apoptosis compared to fresh and overnight IL-2 activated NK cells. Whether expanded NK cells are more susceptible to apoptosis mediated by tumor cells expressing Fas Ligand in vitro and in vivo is currently being investigated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1271.

Nephrotic syndrome associated with thrombotic microangiopathy following allogeneic stem cell transplantation for myelodysplastic syndrome – response to Nakamura et al

We thank Dr Nakamura et al for their comment on our report describing nephrotic syndrome following cyclophosphamide/ fludarabine-based non-myeloablative allogeneic haematopoietic cell transplantation (HCT) (Srinivasan et al, 2005). Nakamura et al describe a patient who developed cyclosporine-associated thrombotic microangiopathy (TMA) and nephrotic syndrome following reduced-intensity HCT. They further suggest that TMA might have contributed to nephrotic syndrome in our patient series. Clinically significant thrombotic thrombocytopenic purpura (TTP)/haemolytic uremic syndrome (HUS), often associated with the use of calcineurin inhibitors, is a well recognised complication of allogeneic HCT (Holler et al, 1989; Rabinowe et al, 1991). Furthermore, proteinuria – including frank nephrotic syndrome – is a well described and not uncommon manifestation of TMA (Magee et al, 2000; Galesic et al, 2006). Therefore, this syndrome should be entertained in the differential diagnosis of nephrotic syndrome in all patients who present with clinical or laboratory features suggestive of TMA (such as thrombocytopenia, haemolysis, neurologic symptoms or renal failure). Six of the seven patients in our series had no clinical evidence of TMA; a renal biopsy was obtained in three of these patients and confirmed a diagnosis of membranous glomerulonephritis without evidence of microangiopathy. One patient (Patient 7) died from a cerebral haemorrhage related to TTP; however, proteinuria preceded the onset of clinical features suggestive of TTP by several months and examination of renal tissue obtained at autopsy revealed incontrovertible evidence of membranous glomerulonephritis in addition to microangiopathic changes. These data suggest that factors other than TMA were responsible for the nephrotic syndrome observed in the cohort of patients described in our report (Srinivasan et al, 2005). Since our original report, we have observed four additional cases of nephrotic syndrome following HCT at our centre. Two of these occurred in patients who received nonmyeloablative conditioning with cyclophosphamide/fludarabine while the other two cases occurred following a T-cell depleted HCT with myeloablative conditioning using cyclophosphamide and total body irradiation. Renal biopsy performed in all four cases showed a variety of renal histopathologic features including membranous glomerulonephritis (one patient), focal segmental glomerulosclerosis (one patient), TMA (one patient) and features suggestive of TMA and a vasculitic process (one patient). It is clear from our experience as well as from previously published reports in patients undergoing myeloablative HCT that nephrotic syndrome in the post-transplant period can result from diverse aetiologies that may require equally diverse management strategies. Consequently, it is our current practice to obtain a histologic diagnosis in every patient who presents with nephrotic syndrome to help guide appropriate management.