Tolga Sutlu | Bogazici University (original) (raw)

Publications by Tolga Sutlu

Journal of extracellular vesicles, 2017

Behçet's disease (BD) activity is characterised by sustained, over-exuberant immune activatio... more Behçet's disease (BD) activity is characterised by sustained, over-exuberant immune activation, yet the underlying mechanisms leading to active BD state are poorly defined. Herein, we show that the human cathelicidin derived antimicrobial peptide LL37 associates with and directs plasma extracellular vesicles (EV) to immune cells, thereby leading to enhanced immune activation aggravating BD pathology. Notably, disease activity was correlated with elevated levels of circulating LL37 and EV plasma concentration. Stimulation of healthy PBMC with active BD patient EVs induced heightened IL1β, IFNα, IL6 and IP10 secretion compared to healthy and inactive BD EVs. Remarkably, when mixed with LL37, healthy plasma-EVs triggered a robust immune activation replicating the pathology inducing properties of BD EVs. The findings of this study could be of clinical interest in the management of BD, implicating LL37/EV association as one of the major contributors of BD pathogenesis. Abbreviations:...

Oncol Rep, 2009

High-dose therapy (HDT) followed by autologous stem cell transplantation (ASCT) is the most commo... more High-dose therapy (HDT) followed by autologous stem cell transplantation (ASCT) is the most common treatment for patients under 65 years of age with multiple myeloma (MM). In this study, we present a retrospective analysis of the prognostic impact of different factors in patients who have received this treatment as first line therapy in our centre. Abnormalities in chromosome 13 were identified by fluorescence in situ hybridization at the time of diagnosis. The median overall survival (OS) and progression-free survival (PFS) from transplantation time in the whole group of 193 patients were 90 and 48 months respectively. The median follow-up was 65 months (range: 6-186 months). The complete remission (CR) rate in patients with and without del(13) was 31 and 40% respectively whereas the median OS in patients with del(13) was 58 months but not reached in patients without del(13) (p=0.006). The PFS was 26 months in patients with del(13) and 84 months in those without del(13) (p=0.001). The transplantation related mortality was 2.5% both in the absence and presence of del(13). Patients who achieved CR following ASCT had longer OS and PFS when compared to those who only achieved partial remission. Thus, this study confirms the role of del(13) as a marker of poor prognosis. Multivariate analysis showed that the existence of del(13) was the only single independent factor effecting survival (p=0.001). In patients without del(13), the prognostic impact was even stronger when combined with the plasma cell load in the bone marrow (p=0.020), whereas the plasma cell load had no effect on survival of patients with del(13). Overall, the absence of del(13) in combination with low plasma cell infiltration at diagnosis predicts the best survival.

PLOS ONE, 2015

Loss of the chromosomal region 8p21 negatively effects survival in patients with multiple myeloma... more Loss of the chromosomal region 8p21 negatively effects survival in patients with multiple myeloma (MM) that undergo autologous stem cell transplantation (ASCT). In this study, we aimed to identify the immunological and molecular consequences of del(8)(p21) with regards to treatment response and bortezomib resistance. In patients receiving bortezomib as a single first line agent without any high-dose therapy, we have observed that patients with del(8)(p21) responded poorly to bortezomib with 50% showing no response while patients without the deletion had a response rate of 90%. In vitro analysis revealed a higher resistance to bortezomib possibly due to an altered gene expression profile caused by del(8)(p21) including genes such as TRAIL-R4, CCDC25, RHOBTB2, PTK2B, SCARA3, MYC, BCL2 and TP53. Furthermore, while bortezomib sensitized MM cells without del(8)(p21) to TRAIL/APO2L mediated apoptosis, in cells with del(8)(p21) bortezomib failed to upregulate the pro-apoptotic death receptors TRAIL-R1 and TRAIL-R2 which are located on the 8p21 region. Also expressing higher levels of the decoy death receptor TRAIL-R4, these cells were largely resistant to TRAIL/APO2L mediated apoptosis. Corroborating the clinical outcome of the patients, our data provides a potential explanation regarding the poor response of MM patients with del(8)(p21) to bortezomib treatment. Furthermore, our clinical analysis suggests that including immunomodulatory agents such as Lenalidomide in the treatment regimen may help to overcome this negative effect, providing an alternative consideration in treatment planning of MM patients with del(8)(p21).

PloS one, 2015

Loss of the chromosomal region 8p21 negatively effects survival in patients with multiple myeloma... more Loss of the chromosomal region 8p21 negatively effects survival in patients with multiple myeloma (MM) that undergo autologous stem cell transplantation (ASCT). In this study, we aimed to identify the immunological and molecular consequences of del(8)(p21) with regards to treatment response and bortezomib resistance. In patients receiving bortezomib as a single first line agent without any high-dose therapy, we have observed that patients with del(8)(p21) responded poorly to bortezomib with 50% showing no response while patients without the deletion had a response rate of 90%. In vitro analysis revealed a higher resistance to bortezomib possibly due to an altered gene expression profile caused by del(8)(p21) including genes such as TRAIL-R4, CCDC25, RHOBTB2, PTK2B, SCARA3, MYC, BCL2 and TP53. Furthermore, while bortezomib sensitized MM cells without del(8)(p21) to TRAIL/APO2L mediated apoptosis, in cells with del(8)(p21) bortezomib failed to upregulate the pro-apoptotic death recept...

Modulation of intracellular signaling pathways or receptor expression in natural killer (NK) cell... more Modulation of intracellular signaling pathways or receptor expression in natural killer (NK) cells by genetic manipulation is an attractive possibility in studies of NK cell specificity and function. Moreover, feasible applications of these genetic manipulations in the context of gene and NK cell therapy regimens may be considered. However, efficient gene modification of primary NK cells has been largely hampered by the absence of an efficient gene-transfer protocol.A retrovirus-based easy-to-use transduction protocol that can insert the gene of interest permanently into primary NK cells would be an important tool to advance our studies in NK cell biology and NK cell-mediated therapies. We have recently described a protocol for efficient expansion of NK cells under good manufacturing practice (GMP) conditions from the healthy donors and from patients with hematological malignancies. As the active division of cells is a prerequisite for efficient retroviral insertion, the high rate of expansion in this protocol provides more efficient transduction by retroviral vectors. We hereby present this simple and efficient retroviral vector-based gene-transfer protocol for such ex vivo cultured primary human NK cells.

Immunotherapy, 2009

The chimeric state after allogeneic hematopoietic stem cell transplantation provides a platform f... more The chimeric state after allogeneic hematopoietic stem cell transplantation provides a platform for adoptive immunotherapy using donor-derived immune cells. The major risk with donor lymphocyte infusions (DLIs) is the development of graft-versus-host disease (GvHD). Development of new DLI products with antitumor reactivity and reduced GvHD risk represents a challenging task in cancer immunotherapy. Although natural killer (NK) and NK-like T cells are promising owing to their antitumor activity, their low concentrations in peripheral blood mononuclear cells reduces their utility in DLIs. We have recently developed a system that allows expansion of clinical-grade NK and NK-like T cells in large numbers. In this study, the safety of donor-derived long-term ex vivo-expanded human NK and NK-like T cells given as DLIs was investigated as immunotherapy for cancer in five patients following allogeneic stem cell infusion. Infusion of the cells was safe whether administered alone or with IL-2 subcutaneously. No signs of acute GvHD were observed. One patient with hepatocellular carcinoma showed markedly decreased serum alpha-fetoprotein levels following cell infusions. These findings suggest that the use of ex vivo-expanded NK and NK-like T cells is safe and appears an attractive approach for further clinical evaluation in cancer patients.

In allogeneic hematopoietic stem cell transplantation, donor-derived T cells are key players for ... more In allogeneic hematopoietic stem cell transplantation, donor-derived T cells are key players for early immune reconstitution and efficient engraftment, as well as the graft-versus-leukemia and graft-versus-infection effects. However, a severe and quite common life-threatening complication is the development of graft-versus-host disease, during which the alloreactive donor T cells attack the host. Controlling graft-versus-host disease while preserving the benefits of graft-versus-leukemia still constitutes a challenge. A promising approach for the control of graft-versus-host disease is suicide gene therapy, which involves the ex vivo genetic modification of donor T cells with a suicide gene that allows for the selective elimination of the cells in vivo if graft-versus-host disease occurs. This article presents an overview of such approaches with special reference to lessons learned from previous clinical experiences, as well as a discussion of critical factors in suicide gene therapy.

Journal of Clinical Investigation, 2014

X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations wi... more X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton's tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTK transcripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we engineered a transgenic mouse that harbors a BAC with an authentic, mutated, splice-defective human BTK gene. BTK transgenic mice were bred onto a Btk knockout background to avoid interference of the orthologous mouse protein. Using this model, we determined that BTK-specific SCOs are able to correct aberrantly spliced BTK in B lymphocytes, including pro-B cells. Correction of BTK mRNA restored expression of functional protein, as shown both by enhanced lymphocyte survival and reestablished BTK activation upon B cell receptor stimulation. Furthermore, SCO treatment corrected splicing and restored BTK expression in primary cells from patients with XLA. Together, our data demonstrate that SCOs can restore BTK function and that BTK-targeting SCOs have potential as personalized medicine in patients with XLA.

The FASEB Journal, 2007

Cell-penetrating peptides (CPPs) are peptides able to promote uptake of various cargos, including... more Cell-penetrating peptides (CPPs) are peptides able to promote uptake of various cargos, including proteins and plasmids. Advances in recent years imply the uptake to be endocytic, where the current hurdle for efficient intracellular delivery is material being retained in the endosomes. In this study we wanted to compare the ability of various established CPPs to deliver siRNA and induce gene silencing of luciferase, with a novel designed penetratin analog having endosomolytic properties, using a noncovalent strategy. In principal, the penetratin analog EB1 will, upon protonation in the early-late endosomes, be able to form an amphipathic alpha helix resulting in permeabilization of the endosomal membrane. We demonstrate that even though all CPPs evaluated in this study can form complexes with siRNA, there is not a direct relationship between the complex formation ability and delivery efficacy. More important, although all CPPs significantly promote siRNA uptake, in some cases no gene silencing effect can be observed unless endosomal escape is induced. We find the designed endosomolytic peptide EB1 to be far more effective both in forming complexes and transporting biologically active siRNA than its parent peptide penetratin. We believe that developing CPPs with increased endosomolytical properties is a necessary step toward achieving biological effects at low concentrations for future in vivo applications.

Oncology Reports, 2009

High-dose therapy (HDT) followed by autologous stem cell transplantation (ASCT) is the most commo... more High-dose therapy (HDT) followed by autologous stem cell transplantation (ASCT) is the most common treatment for patients under 65 years of age with multiple myeloma (MM). In this study, we present a retrospective analysis of the prognostic impact of different factors in patients who have received this treatment as first line therapy in our centre. Abnormalities in chromosome 13 were identified by fluorescence in situ hybridization at the time of diagnosis. The median overall survival (OS) and progression-free survival (PFS) from transplantation time in the whole group of 193 patients were 90 and 48 months respectively. The median follow-up was 65 months (range: 6-186 months). The complete remission (CR) rate in patients with and without del(13) was 31 and 40% respectively whereas the median OS in patients with del(13) was 58 months but not reached in patients without del(13) (p=0.006). The PFS was 26 months in patients with del(13) and 84 months in those without del(13) (p=0.001). The transplantation related mortality was 2.5% both in the absence and presence of del(13). Patients who achieved CR following ASCT had longer OS and PFS when compared to those who only achieved partial remission. Thus, this study confirms the role of del(13) as a marker of poor prognosis. Multivariate analysis showed that the existence of del(13) was the only single independent factor effecting survival (p=0.001). In patients without del(13), the prognostic impact was even stronger when combined with the plasma cell load in the bone marrow (p=0.020), whereas the plasma cell load had no effect on survival of patients with del(13). Overall, the absence of del(13) in combination with low plasma cell infiltration at diagnosis predicts the best survival.

Leukemia Research, 2010

The constitutional pericentric inversion on chromosome 3, inv(3), is rarely found in a normal pop... more The constitutional pericentric inversion on chromosome 3, inv(3), is rarely found in a normal population. The aim of our study was to investigate its possible link to hematologic malignancy. Chromosomes from bone marrow cells in 890 patients with hematologic disorders were analyzed with the Q-banding technique. Thirty-four patients had inv(3) (3.8%). In 241 patients with myelodysplastic syndromes the frequency was 6.2% as opposed to 2.9% in the remaining 649 patients (p = 0.02). The increased frequency of inv(3) in patients with myelodysplastic syndromes indicates that inv(3) could be a risk factor for the development of the disease.

Journal of Internal Medicine, 2009

Sutlu T, Alici E (Karolinska Institutet, Stockholm, Sweden). Natural killer cell-based immunother... more Sutlu T, Alici E (Karolinska Institutet, Stockholm, Sweden). Natural killer cell-based immunotherapy in cancer: current insights and future prospects (Review). J Intern Med 2009; 266: 154-181.

Journal of Internal Medicine, 2011

Nahi H., Sutlu T., Jansson M., Alici E., Gahrton G. (

Journal of Biological Chemistry, 2005

A novel gene sequence, with two exons and one intron, encoding a metallothionein (MT) has been id... more A novel gene sequence, with two exons and one intron, encoding a metallothionein (MT) has been identified in durum wheat Triticum durum cv. Balcali85 genomic DNA. Multiple alignment analyses on the cDNA and the translated protein sequences showed that T. durum MT (dMT) can be classified as a type 1 MT. dMT has three Cys-X-Cys motifs in each of the Nand C-terminal domains and a 42-residue-long hinge region devoid of cysteines. dMT was overexpressed in Escherichia coli as a fusion protein (GSTdMT), and bacteria expressing the fusion protein showed increased tolerance to cadmium in the growth medium compared with controls. Purified GSTdMT was characterized by SDS-and native-PAGE, size exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. It was shown that the recombinant protein binds 4 ؎ 1 mol of cadmium/mol of protein and has a high tendency to form stable oligomeric structures. The structure of GSTdMT and dMT was investigated by synchrotron x-ray solution scattering and computational methods. X-ray scattering measurements indicated a strong tendency for GSTdMT to form dimers and trimers in solution and yielded structural models that were compatible with a stable dimeric form in which dMT had an extended conformation. Results of homology modeling and ab initio solution scattering approaches produced an elongated dMT structure with a long central hinge region. The predicted model and those obtained from x-ray scattering are in agreement and suggest that dMT may be involved in functions other than metal detoxification. The abbreviations used are: MT, metallothionein, dmt; T. durum metallothionein gene; amt, T. aestivum metallothionein gene; RT, reverse transcriptase; GST, glutathione S-transferase; dMT, T. durum metallothionein; GSTdMT, fusion protein of GST and dMT, MALDI-TOF-MS, matrix-assisted laser desorption/ionization time of flight mass spectrometry; Ec, Early cysteine labeled; MM, molecular mass; MOPS, 4-morpholinepropanesulfonic acid.

Human Gene Therapy, 2012

Adoptive immunotherapy with genetically modified natural killer (NK) cells is a promising approac... more Adoptive immunotherapy with genetically modified natural killer (NK) cells is a promising approach for cancer treatment. Yet, optimization of highly efficient and clinically applicable gene transfer protocols for NK cells still presents a challenge. In this study, we aimed at identifying conditions under which optimum lentiviral gene transfer to NK cells can be achieved. Our results demonstrate that stimulation of NK cells with interleukin (IL)-2 and IL-21 supports efficient transduction using a VSV-G pseudotyped lentiviral vector. Moreover, we have identified that inhibition of innate immune receptor signaling greatly enhances transduction efficiency. We were able to boost the efficiency of lentiviral genetic modification on average 3.8-fold using BX795, an inhibitor of the TBK1/IKKe complex acting downstream of RIG-I, MDA-5, and TLR3. We have also observed that the use of BX795 enhances lentiviral transduction efficiency in a number of human and mouse cell lines, indicating a broadly applicable, practical, and safe approach that has the potential of being applicable to various gene therapy protocols.

Experimental Hematology, 2007

Objective. Despite advances in autologous stem cell transplantation and chemotherapy, multiple my... more Objective. Despite advances in autologous stem cell transplantation and chemotherapy, multiple myeloma (MM) remains an incurable disease. Due to the role of natural killer (NK) cells in host resistance against several tumors, it is of interest to explore the anti-MM activity of NK cells. For this reason, we aimed to determine if NK cells provide anti-MM activity following interleukin-2 (IL-2) administration, and if ex vivo activated and intravenously administered NK cells prolong survival in MM-bearing C57BL/KaLwRij mice. Methods. The anti-MM effect of IL-2 was tested by intraperitoneal injection into the 5T33MM-inoculated mice. Subsequently, in vivo effector cell depletions were performed by administration of anti-NK1.1 or anti-CD8 monoclonal antibodies. Finally, magnetically separated and activated NK cells from splenocytes of C57BL/KaLwRij mice were adoptively transferred to tumor-bearing mice in conjunction with IL-2 treatment. Results. IL-2 administration into MM-bearing mice significantly prolonged their survival. This effect was diminished by in vivo depletion of NK cells. Adoptive transfer of activated NK cells showed a significant in vivo anti-MM effect that was dependent on cell dose. Biodistribution of the marked adoptively transferred NK cells correlated with MM cells' homing sites. Conclusion. These data suggest that activated NK cells have a promising potential in adoptive immunotherapy for MM. Ó

Cytotherapy, 2010

Natural killer (NK) cell-based adoptive immunotherapy is a promising approach for the treatment o... more Natural killer (NK) cell-based adoptive immunotherapy is a promising approach for the treatment of cancer. Ex vivo expansion and activation of NK cells under good manufacturing practice (GMP) conditions are crucial for facilitating large clinical trials. The goal of this study was to optimize a large-scale, feeder-free, closed system for efficient NK cell expansion. Peripheral blood mononuclear cells (PBMCs) from healthy donors and myeloma patients were cultured for 21 days using flasks, cell culture bags and bioreactors. Final products from different expansions were evaluated comparatively for phenotype and functionality. Significant NK cell expansions were obtained in all systems. The bioreactor yielded a final product rich in NK cells (mean 38%) ensuring that a clinically relevant cell dose was reached (mean 9.8 x 10⁹ NK cells). Moreover, we observed that NK cells expanded in the bioreactor displayed significantly higher cytotoxic capacity. It was possible to attribute this partially to a higher expression level of NKp44 compared with NK cells expanded in flasks. These results demonstrate that large amounts of highly active NK cells for adoptive immunotherapy can be produced in a closed, automated, large-scale bioreactor under feeder-free current GMP conditions, facilitating clinical trials for the use of these cells.

Journal of extracellular vesicles, 2017

Behçet's disease (BD) activity is characterised by sustained, over-exuberant immune activatio... more Behçet's disease (BD) activity is characterised by sustained, over-exuberant immune activation, yet the underlying mechanisms leading to active BD state are poorly defined. Herein, we show that the human cathelicidin derived antimicrobial peptide LL37 associates with and directs plasma extracellular vesicles (EV) to immune cells, thereby leading to enhanced immune activation aggravating BD pathology. Notably, disease activity was correlated with elevated levels of circulating LL37 and EV plasma concentration. Stimulation of healthy PBMC with active BD patient EVs induced heightened IL1β, IFNα, IL6 and IP10 secretion compared to healthy and inactive BD EVs. Remarkably, when mixed with LL37, healthy plasma-EVs triggered a robust immune activation replicating the pathology inducing properties of BD EVs. The findings of this study could be of clinical interest in the management of BD, implicating LL37/EV association as one of the major contributors of BD pathogenesis. Abbreviations:...

Oncol Rep, 2009

High-dose therapy (HDT) followed by autologous stem cell transplantation (ASCT) is the most commo... more High-dose therapy (HDT) followed by autologous stem cell transplantation (ASCT) is the most common treatment for patients under 65 years of age with multiple myeloma (MM). In this study, we present a retrospective analysis of the prognostic impact of different factors in patients who have received this treatment as first line therapy in our centre. Abnormalities in chromosome 13 were identified by fluorescence in situ hybridization at the time of diagnosis. The median overall survival (OS) and progression-free survival (PFS) from transplantation time in the whole group of 193 patients were 90 and 48 months respectively. The median follow-up was 65 months (range: 6-186 months). The complete remission (CR) rate in patients with and without del(13) was 31 and 40% respectively whereas the median OS in patients with del(13) was 58 months but not reached in patients without del(13) (p=0.006). The PFS was 26 months in patients with del(13) and 84 months in those without del(13) (p=0.001). The transplantation related mortality was 2.5% both in the absence and presence of del(13). Patients who achieved CR following ASCT had longer OS and PFS when compared to those who only achieved partial remission. Thus, this study confirms the role of del(13) as a marker of poor prognosis. Multivariate analysis showed that the existence of del(13) was the only single independent factor effecting survival (p=0.001). In patients without del(13), the prognostic impact was even stronger when combined with the plasma cell load in the bone marrow (p=0.020), whereas the plasma cell load had no effect on survival of patients with del(13). Overall, the absence of del(13) in combination with low plasma cell infiltration at diagnosis predicts the best survival.

PLOS ONE, 2015

Loss of the chromosomal region 8p21 negatively effects survival in patients with multiple myeloma... more Loss of the chromosomal region 8p21 negatively effects survival in patients with multiple myeloma (MM) that undergo autologous stem cell transplantation (ASCT). In this study, we aimed to identify the immunological and molecular consequences of del(8)(p21) with regards to treatment response and bortezomib resistance. In patients receiving bortezomib as a single first line agent without any high-dose therapy, we have observed that patients with del(8)(p21) responded poorly to bortezomib with 50% showing no response while patients without the deletion had a response rate of 90%. In vitro analysis revealed a higher resistance to bortezomib possibly due to an altered gene expression profile caused by del(8)(p21) including genes such as TRAIL-R4, CCDC25, RHOBTB2, PTK2B, SCARA3, MYC, BCL2 and TP53. Furthermore, while bortezomib sensitized MM cells without del(8)(p21) to TRAIL/APO2L mediated apoptosis, in cells with del(8)(p21) bortezomib failed to upregulate the pro-apoptotic death receptors TRAIL-R1 and TRAIL-R2 which are located on the 8p21 region. Also expressing higher levels of the decoy death receptor TRAIL-R4, these cells were largely resistant to TRAIL/APO2L mediated apoptosis. Corroborating the clinical outcome of the patients, our data provides a potential explanation regarding the poor response of MM patients with del(8)(p21) to bortezomib treatment. Furthermore, our clinical analysis suggests that including immunomodulatory agents such as Lenalidomide in the treatment regimen may help to overcome this negative effect, providing an alternative consideration in treatment planning of MM patients with del(8)(p21).

PloS one, 2015

Loss of the chromosomal region 8p21 negatively effects survival in patients with multiple myeloma... more Loss of the chromosomal region 8p21 negatively effects survival in patients with multiple myeloma (MM) that undergo autologous stem cell transplantation (ASCT). In this study, we aimed to identify the immunological and molecular consequences of del(8)(p21) with regards to treatment response and bortezomib resistance. In patients receiving bortezomib as a single first line agent without any high-dose therapy, we have observed that patients with del(8)(p21) responded poorly to bortezomib with 50% showing no response while patients without the deletion had a response rate of 90%. In vitro analysis revealed a higher resistance to bortezomib possibly due to an altered gene expression profile caused by del(8)(p21) including genes such as TRAIL-R4, CCDC25, RHOBTB2, PTK2B, SCARA3, MYC, BCL2 and TP53. Furthermore, while bortezomib sensitized MM cells without del(8)(p21) to TRAIL/APO2L mediated apoptosis, in cells with del(8)(p21) bortezomib failed to upregulate the pro-apoptotic death recept...

Modulation of intracellular signaling pathways or receptor expression in natural killer (NK) cell... more Modulation of intracellular signaling pathways or receptor expression in natural killer (NK) cells by genetic manipulation is an attractive possibility in studies of NK cell specificity and function. Moreover, feasible applications of these genetic manipulations in the context of gene and NK cell therapy regimens may be considered. However, efficient gene modification of primary NK cells has been largely hampered by the absence of an efficient gene-transfer protocol.A retrovirus-based easy-to-use transduction protocol that can insert the gene of interest permanently into primary NK cells would be an important tool to advance our studies in NK cell biology and NK cell-mediated therapies. We have recently described a protocol for efficient expansion of NK cells under good manufacturing practice (GMP) conditions from the healthy donors and from patients with hematological malignancies. As the active division of cells is a prerequisite for efficient retroviral insertion, the high rate of expansion in this protocol provides more efficient transduction by retroviral vectors. We hereby present this simple and efficient retroviral vector-based gene-transfer protocol for such ex vivo cultured primary human NK cells.

Immunotherapy, 2009

The chimeric state after allogeneic hematopoietic stem cell transplantation provides a platform f... more The chimeric state after allogeneic hematopoietic stem cell transplantation provides a platform for adoptive immunotherapy using donor-derived immune cells. The major risk with donor lymphocyte infusions (DLIs) is the development of graft-versus-host disease (GvHD). Development of new DLI products with antitumor reactivity and reduced GvHD risk represents a challenging task in cancer immunotherapy. Although natural killer (NK) and NK-like T cells are promising owing to their antitumor activity, their low concentrations in peripheral blood mononuclear cells reduces their utility in DLIs. We have recently developed a system that allows expansion of clinical-grade NK and NK-like T cells in large numbers. In this study, the safety of donor-derived long-term ex vivo-expanded human NK and NK-like T cells given as DLIs was investigated as immunotherapy for cancer in five patients following allogeneic stem cell infusion. Infusion of the cells was safe whether administered alone or with IL-2 subcutaneously. No signs of acute GvHD were observed. One patient with hepatocellular carcinoma showed markedly decreased serum alpha-fetoprotein levels following cell infusions. These findings suggest that the use of ex vivo-expanded NK and NK-like T cells is safe and appears an attractive approach for further clinical evaluation in cancer patients.

In allogeneic hematopoietic stem cell transplantation, donor-derived T cells are key players for ... more In allogeneic hematopoietic stem cell transplantation, donor-derived T cells are key players for early immune reconstitution and efficient engraftment, as well as the graft-versus-leukemia and graft-versus-infection effects. However, a severe and quite common life-threatening complication is the development of graft-versus-host disease, during which the alloreactive donor T cells attack the host. Controlling graft-versus-host disease while preserving the benefits of graft-versus-leukemia still constitutes a challenge. A promising approach for the control of graft-versus-host disease is suicide gene therapy, which involves the ex vivo genetic modification of donor T cells with a suicide gene that allows for the selective elimination of the cells in vivo if graft-versus-host disease occurs. This article presents an overview of such approaches with special reference to lessons learned from previous clinical experiences, as well as a discussion of critical factors in suicide gene therapy.

Journal of Clinical Investigation, 2014

X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations wi... more X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton's tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTK transcripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we engineered a transgenic mouse that harbors a BAC with an authentic, mutated, splice-defective human BTK gene. BTK transgenic mice were bred onto a Btk knockout background to avoid interference of the orthologous mouse protein. Using this model, we determined that BTK-specific SCOs are able to correct aberrantly spliced BTK in B lymphocytes, including pro-B cells. Correction of BTK mRNA restored expression of functional protein, as shown both by enhanced lymphocyte survival and reestablished BTK activation upon B cell receptor stimulation. Furthermore, SCO treatment corrected splicing and restored BTK expression in primary cells from patients with XLA. Together, our data demonstrate that SCOs can restore BTK function and that BTK-targeting SCOs have potential as personalized medicine in patients with XLA.

The FASEB Journal, 2007

Cell-penetrating peptides (CPPs) are peptides able to promote uptake of various cargos, including... more Cell-penetrating peptides (CPPs) are peptides able to promote uptake of various cargos, including proteins and plasmids. Advances in recent years imply the uptake to be endocytic, where the current hurdle for efficient intracellular delivery is material being retained in the endosomes. In this study we wanted to compare the ability of various established CPPs to deliver siRNA and induce gene silencing of luciferase, with a novel designed penetratin analog having endosomolytic properties, using a noncovalent strategy. In principal, the penetratin analog EB1 will, upon protonation in the early-late endosomes, be able to form an amphipathic alpha helix resulting in permeabilization of the endosomal membrane. We demonstrate that even though all CPPs evaluated in this study can form complexes with siRNA, there is not a direct relationship between the complex formation ability and delivery efficacy. More important, although all CPPs significantly promote siRNA uptake, in some cases no gene silencing effect can be observed unless endosomal escape is induced. We find the designed endosomolytic peptide EB1 to be far more effective both in forming complexes and transporting biologically active siRNA than its parent peptide penetratin. We believe that developing CPPs with increased endosomolytical properties is a necessary step toward achieving biological effects at low concentrations for future in vivo applications.

Oncology Reports, 2009

High-dose therapy (HDT) followed by autologous stem cell transplantation (ASCT) is the most commo... more High-dose therapy (HDT) followed by autologous stem cell transplantation (ASCT) is the most common treatment for patients under 65 years of age with multiple myeloma (MM). In this study, we present a retrospective analysis of the prognostic impact of different factors in patients who have received this treatment as first line therapy in our centre. Abnormalities in chromosome 13 were identified by fluorescence in situ hybridization at the time of diagnosis. The median overall survival (OS) and progression-free survival (PFS) from transplantation time in the whole group of 193 patients were 90 and 48 months respectively. The median follow-up was 65 months (range: 6-186 months). The complete remission (CR) rate in patients with and without del(13) was 31 and 40% respectively whereas the median OS in patients with del(13) was 58 months but not reached in patients without del(13) (p=0.006). The PFS was 26 months in patients with del(13) and 84 months in those without del(13) (p=0.001). The transplantation related mortality was 2.5% both in the absence and presence of del(13). Patients who achieved CR following ASCT had longer OS and PFS when compared to those who only achieved partial remission. Thus, this study confirms the role of del(13) as a marker of poor prognosis. Multivariate analysis showed that the existence of del(13) was the only single independent factor effecting survival (p=0.001). In patients without del(13), the prognostic impact was even stronger when combined with the plasma cell load in the bone marrow (p=0.020), whereas the plasma cell load had no effect on survival of patients with del(13). Overall, the absence of del(13) in combination with low plasma cell infiltration at diagnosis predicts the best survival.

Leukemia Research, 2010

The constitutional pericentric inversion on chromosome 3, inv(3), is rarely found in a normal pop... more The constitutional pericentric inversion on chromosome 3, inv(3), is rarely found in a normal population. The aim of our study was to investigate its possible link to hematologic malignancy. Chromosomes from bone marrow cells in 890 patients with hematologic disorders were analyzed with the Q-banding technique. Thirty-four patients had inv(3) (3.8%). In 241 patients with myelodysplastic syndromes the frequency was 6.2% as opposed to 2.9% in the remaining 649 patients (p = 0.02). The increased frequency of inv(3) in patients with myelodysplastic syndromes indicates that inv(3) could be a risk factor for the development of the disease.

Journal of Internal Medicine, 2009

Sutlu T, Alici E (Karolinska Institutet, Stockholm, Sweden). Natural killer cell-based immunother... more Sutlu T, Alici E (Karolinska Institutet, Stockholm, Sweden). Natural killer cell-based immunotherapy in cancer: current insights and future prospects (Review). J Intern Med 2009; 266: 154-181.

Journal of Internal Medicine, 2011

Nahi H., Sutlu T., Jansson M., Alici E., Gahrton G. (

Journal of Biological Chemistry, 2005

A novel gene sequence, with two exons and one intron, encoding a metallothionein (MT) has been id... more A novel gene sequence, with two exons and one intron, encoding a metallothionein (MT) has been identified in durum wheat Triticum durum cv. Balcali85 genomic DNA. Multiple alignment analyses on the cDNA and the translated protein sequences showed that T. durum MT (dMT) can be classified as a type 1 MT. dMT has three Cys-X-Cys motifs in each of the Nand C-terminal domains and a 42-residue-long hinge region devoid of cysteines. dMT was overexpressed in Escherichia coli as a fusion protein (GSTdMT), and bacteria expressing the fusion protein showed increased tolerance to cadmium in the growth medium compared with controls. Purified GSTdMT was characterized by SDS-and native-PAGE, size exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. It was shown that the recombinant protein binds 4 ؎ 1 mol of cadmium/mol of protein and has a high tendency to form stable oligomeric structures. The structure of GSTdMT and dMT was investigated by synchrotron x-ray solution scattering and computational methods. X-ray scattering measurements indicated a strong tendency for GSTdMT to form dimers and trimers in solution and yielded structural models that were compatible with a stable dimeric form in which dMT had an extended conformation. Results of homology modeling and ab initio solution scattering approaches produced an elongated dMT structure with a long central hinge region. The predicted model and those obtained from x-ray scattering are in agreement and suggest that dMT may be involved in functions other than metal detoxification. The abbreviations used are: MT, metallothionein, dmt; T. durum metallothionein gene; amt, T. aestivum metallothionein gene; RT, reverse transcriptase; GST, glutathione S-transferase; dMT, T. durum metallothionein; GSTdMT, fusion protein of GST and dMT, MALDI-TOF-MS, matrix-assisted laser desorption/ionization time of flight mass spectrometry; Ec, Early cysteine labeled; MM, molecular mass; MOPS, 4-morpholinepropanesulfonic acid.

Human Gene Therapy, 2012

Adoptive immunotherapy with genetically modified natural killer (NK) cells is a promising approac... more Adoptive immunotherapy with genetically modified natural killer (NK) cells is a promising approach for cancer treatment. Yet, optimization of highly efficient and clinically applicable gene transfer protocols for NK cells still presents a challenge. In this study, we aimed at identifying conditions under which optimum lentiviral gene transfer to NK cells can be achieved. Our results demonstrate that stimulation of NK cells with interleukin (IL)-2 and IL-21 supports efficient transduction using a VSV-G pseudotyped lentiviral vector. Moreover, we have identified that inhibition of innate immune receptor signaling greatly enhances transduction efficiency. We were able to boost the efficiency of lentiviral genetic modification on average 3.8-fold using BX795, an inhibitor of the TBK1/IKKe complex acting downstream of RIG-I, MDA-5, and TLR3. We have also observed that the use of BX795 enhances lentiviral transduction efficiency in a number of human and mouse cell lines, indicating a broadly applicable, practical, and safe approach that has the potential of being applicable to various gene therapy protocols.

Experimental Hematology, 2007

Objective. Despite advances in autologous stem cell transplantation and chemotherapy, multiple my... more Objective. Despite advances in autologous stem cell transplantation and chemotherapy, multiple myeloma (MM) remains an incurable disease. Due to the role of natural killer (NK) cells in host resistance against several tumors, it is of interest to explore the anti-MM activity of NK cells. For this reason, we aimed to determine if NK cells provide anti-MM activity following interleukin-2 (IL-2) administration, and if ex vivo activated and intravenously administered NK cells prolong survival in MM-bearing C57BL/KaLwRij mice. Methods. The anti-MM effect of IL-2 was tested by intraperitoneal injection into the 5T33MM-inoculated mice. Subsequently, in vivo effector cell depletions were performed by administration of anti-NK1.1 or anti-CD8 monoclonal antibodies. Finally, magnetically separated and activated NK cells from splenocytes of C57BL/KaLwRij mice were adoptively transferred to tumor-bearing mice in conjunction with IL-2 treatment. Results. IL-2 administration into MM-bearing mice significantly prolonged their survival. This effect was diminished by in vivo depletion of NK cells. Adoptive transfer of activated NK cells showed a significant in vivo anti-MM effect that was dependent on cell dose. Biodistribution of the marked adoptively transferred NK cells correlated with MM cells' homing sites. Conclusion. These data suggest that activated NK cells have a promising potential in adoptive immunotherapy for MM. Ó

Cytotherapy, 2010

Natural killer (NK) cell-based adoptive immunotherapy is a promising approach for the treatment o... more Natural killer (NK) cell-based adoptive immunotherapy is a promising approach for the treatment of cancer. Ex vivo expansion and activation of NK cells under good manufacturing practice (GMP) conditions are crucial for facilitating large clinical trials. The goal of this study was to optimize a large-scale, feeder-free, closed system for efficient NK cell expansion. Peripheral blood mononuclear cells (PBMCs) from healthy donors and myeloma patients were cultured for 21 days using flasks, cell culture bags and bioreactors. Final products from different expansions were evaluated comparatively for phenotype and functionality. Significant NK cell expansions were obtained in all systems. The bioreactor yielded a final product rich in NK cells (mean 38%) ensuring that a clinically relevant cell dose was reached (mean 9.8 x 10⁹ NK cells). Moreover, we observed that NK cells expanded in the bioreactor displayed significantly higher cytotoxic capacity. It was possible to attribute this partially to a higher expression level of NKp44 compared with NK cells expanded in flasks. These results demonstrate that large amounts of highly active NK cells for adoptive immunotherapy can be produced in a closed, automated, large-scale bioreactor under feeder-free current GMP conditions, facilitating clinical trials for the use of these cells.