Ece Canan Sayitoglu

All Publications

• Engineered Type 1 Regulatory T Cells Have a Cytotoxic Profile and Kill Pediatric Acute Myeloid Leukemia Cells Sayitoglu, E., Uyeda, M., Liu, J. M., Cieniewicz, B., Chen, P., Lacayo, N., Cepika, A., Roncarolo, M. CELL PRESS. 2021: 317

  View details for Web of Science ID 000645188700642

• Pre-clinical development and molecular characterization of an engineered type 1 regulatory T-cell product suitable for immunotherapy. Cytotherapy Liu, J. M., Chen, P., Uyeda, M. J., Cieniewicz, B., Sayitoglu, E. C., Thomas, B. C., Sato, Y., Bacchetta, R., Cepika, A. M., Roncarolo, M. G. 2021

  Abstract

  Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapeutic approach for many hematological disorders. However, allo-HSCT is frequently accompanied by a serious side effect: graft-versus-host disease (GVHD). The clinical use of allo-HSCT is limited by the inability of current immunosuppressive regimens to adequately control GvHD without impairing the graft-versus-leukemia effect (GvL) conferred by transplanted healthy immune cells. To address this, the authors have developed an engineered type 1 regulatory T-cell product called CD4IL-10 cells. CD4IL-10 cells are obtained through lentiviral transduction, which delivers the human IL10 gene into purified polyclonal CD4+ T cells. CD4IL-10 cells may provide an advantage over standard-of-care immunosuppressants because of the ability to suppress GvHD through continuous secretion of IL-10 and enhance the GvL effect in myeloid malignancies through targeted killing of malignant myeloid cells.Here the authors established a production process aimed at current Good Manufacturing Practice (cGMP) production for CD4IL-10 cells.The authors demonstrated that the CD4IL-10 cell product maintains the suppressive and cytotoxic functions of previously described CD4IL-10 cells. In addition, RNA sequencing analysis of CD4IL-10 identified novel transcriptome changes, indicating that CD4IL-10 cells primarily upregulate cytotoxicity-related genes. These include four molecules with described roles in CD8+ T and natural killer cell-mediated cytotoxicity: CD244, KLRD1, KLRC1 and FASLG. Finally, it was shown that CD4IL-10 cells upregulate IL-22, which mediates wound healing and tissue repair, particularly in the gut.Collectively, these results pave the way toward clinical translation of the cGMP-optimized CD4IL-10 cell product and uncover new molecules that have a role in the clinical application of CD4IL-10 cells.

  View details for DOI 10.1016/j.jcyt.2021.05.010

  View details for PubMedID 34404616

• The Yin and Yang of Type 1 Regulatory T Cells: From Discovery to Clinical Application. Frontiers in immunology Sayitoglu, E. C., Freeborn, R. A., Roncarolo, M. G. 2021; 12: 693105

  Abstract

  Regulatory T cells are essential players of peripheral tolerance and suppression of inflammatory immune responses. Type 1 regulatory T (Tr1) cells are FoxP3- regulatory T cells induced in the periphery under tolerogenic conditions. Tr1 cells are identified as LAG3+CD49b+ mature CD4+ T cells that promote peripheral tolerance through secretion of IL-10 and TGF-beta in addition to exerting perforin- and granzyme B-mediated cytotoxicity against myeloid cells. After the initial challenges of isolation were overcome by surface marker identification, ex vivo expansion of antigen-specific Tr1 cells in the presence of tolerogenic dendritic cells (DCs) and IL-10 paved the way for their use in clinical trials. With one Tr1-enriched cell therapy product already in a Phase I clinical trial in the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT), Tr1 cell therapy demonstrates promising results so far in terms of efficacy and safety. In the current review, we identify developments in phenotypic and molecular characterization of Tr1 cells and discuss the potential of engineered Tr1-like cells for clinical applications of Tr1 cell therapies. More than 3 decades after their initial discovery, Tr1 cell therapy is now being used to prevent graft versus host disease (GvHD) in allo-HSCT and will be an alternative to immunosuppression to promote graft tolerance in solid organ transplantation in the near future.

  View details for DOI 10.3389/fimmu.2021.693105

  View details for PubMedID 34177953

• Characterization of zika virus infection of human fetal cardiac mesenchymal stromal cells. PloS one Rossi, F., Josey, B., Sayitoglu, E. C., Potens, R., Sultu, T., Duru, A. D., Beljanski, V. 2020; 15 (9): e0239238

  Abstract

  Zika virus (ZIKV) is a single-stranded RNA virus belonging to the family Flaviviridae. ZIKV predominantly enters cells using the TAM-family protein tyrosine kinase receptor AXL, which is expressed on a range of cell types, including neural progenitor cells, keratinocytes, dendritic cells, and osteoblasts. ZIKV infections have been associated with fetal brain damage, which prompted the World Health Organization to declare a public health emergency in 2016. ZIKV infection has also been linked to birth defects in other organs. Several studies have reported congenital heart defects (CHD) in ZIKV infected infants and cardiovascular complications in adults infected with ZIKV. To develop a better understanding of potential causes for these pathologies at a cellular level, we characterized ZIKV infection of human fetal cardiac mesenchymal stromal cells (fcMSCs), a cell type that is known to contribute to both embryological development as well as adult cardiac physiology. Total RNA, supernatants, and/or cells were collected at various time points post-infection to evaluate ZIKV replication, cell death, and antiviral responses. We found that ZIKV productively infected fcMSCs with peak (~70%) viral mRNA detected at 48 h. Use of an antibody blocking the AXL receptor decreased ZIKV infection (by ~50%), indicating that the receptor is responsible to a large extent for viral entry into the cell. ZIKV also altered protein expression of several mesenchymal cell markers, which suggests that ZIKV could affect fcMSCs' differentiation process. Gene expression analysis of fcMSCs exposed to ZIKV at 6, 12, and 24 h post-infection revealed up-regulation of genes/pathways associated with interferon-stimulated antiviral responses. Stimulation of TLR3 (using poly I:C) or TLR7 (using Imiquimod) prior to ZIKV infection suppressed viral replication in a dose-dependent manner. Overall, fcMSCs can be a target for ZIKV infection, potentially resulting in CHD during embryological development and/or cardiovascular issues in ZIKV infected adults.

  View details for DOI 10.1371/journal.pone.0239238

  View details for PubMedID 32941515

• Boosting Natural Killer Cell-Mediated Targeting of Sarcoma Through DNAM-1 and NKG2D. Frontiers in immunology Sayitoglu, E. C., Georgoudaki, A. M., Chrobok, M., Ozkazanc, D., Josey, B. J., Arif, M., Kusser, K., Hartman, M., Chinn, T. M., Potens, R., Pamukcu, C., Krueger, R., Zhang, C., Mardinoglu, A., Alici, E., Temple, H. T., Sutlu, T., Duru, A. D. 2020; 11: 40

  Abstract

  Sarcomas are malignancies of mesenchymal origin that occur in bone and soft tissues. Many are chemo- and radiotherapy resistant, thus conventional treatments fail to increase overall survival. Natural Killer (NK) cells exert anti-tumor activity upon detection of a complex array of tumor ligands, but this has not been thoroughly explored in the context of sarcoma immunotherapy. In this study, we investigated the NK cell receptor/ligand immune profile of primary human sarcoma explants. Analysis of tumors from 32 sarcoma patients identified the proliferative marker PCNA and DNAM-1 ligands CD112 and/or CD155 as commonly expressed antigens that could be efficiently targeted by genetically modified (GM) NK cells. Despite the strong expression of CD112 and CD155 on sarcoma cells, characterization of freshly dissociated sarcomas revealed a general decrease in tumor-infiltrating NK cells compared to the periphery, suggesting a defect in the endogenous NK cell response. We also applied a functional screening approach to identify relevant NK cell receptor/ligand interactions that induce efficient anti-tumor responses using a panel NK-92 cell lines GM to over-express 12 different activating receptors. Using GM NK-92 cells against primary sarcoma explants (n = 12) revealed that DNAM-1 over-expression on NK-92 cells led to efficient degranulation against all tested explants (n = 12). Additionally, NKG2D over-expression showed enhanced responses against 10 out of 12 explants. These results show that DNAM-1+ or NKG2D+ GM NK-92 cells may be an efficient approach in targeting sarcomas. The degranulation capacity of GM NK-92 cell lines was also tested against various established tumor cell lines, including neuroblastoma, Schwannoma, melanoma, myeloma, leukemia, prostate, pancreatic, colon, and lung cancer. Enhanced degranulation of DNAM-1+ or NKG2D+ GM NK-92 cells was observed against the majority of tumor cell lines tested. In conclusion, DNAM-1 or NKG2D over-expression elicited a dynamic increase in NK cell degranulation against all sarcoma explants and cancer cell lines tested, including those that failed to induce a notable response in WT NK-92 cells. These results support the broad therapeutic potential of DNAM-1+ or NKG2D+ GM NK-92 cells and GM human NK cells for the treatment of sarcomas and other malignancies.

  View details for DOI 10.3389/fimmu.2020.00040

  View details for PubMedID 32082316

  View details for PubMedCentralID PMC7001093

• Engineering antigen-specific NK cell lines against the melanoma-associated antigen tyrosinase via TCR gene transfer EUROPEAN JOURNAL OF IMMUNOLOGY Parlar, A., Sayitoglu, E., Ozkazanc, D., Georgoudaki, A., Pamukcu, C., Aras, M., Josey, B. J., Chrobok, M., Branecki, S., Zahedimaram, P., Ikromzoda, L., Alici, E., Erman, B., Duru, A. D., Sutlu, T. 2019; 49 (8): 1278–90

  Abstract

  Introduction of Chimeric Antigen Receptors to NK cells has so far been the main practical method for targeting NK cells to specific surface antigens. In contrast, T cell receptor (TCR) gene delivery can supply large populations of cytotoxic T-lymphocytes (CTL) targeted against intracellular antigens. However, a major barrier in the development of safe CTL-TCR therapies exists, wherein the mispairing of endogenous and genetically transferred TCR subunits leads to formation of TCRs with off-target specificity. To overcome this and enable specific intracellular antigen targeting, we have tested the use of NK cells for TCR gene transfer to human cells. Our results show that ectopic expression of TCR α/β chains, along with CD3 subunits, enables the functional expression of an antigen-specific TCR complex on NK cell lines NK-92 and YTS, demonstrated by using a TCR against the HLA-A2-restricted tyrosinase-derived melanoma epitope, Tyr368-377 . Most importantly, the introduction of a TCR complex to NK cell lines enables MHC-restricted, antigen-specific killing of tumor cells both in vitro and in vivo. Targeting of NK cells via TCR gene delivery stands out as a novel tool in the field of adoptive immunotherapy which can also overcome the major hurdle of "mispairing" in TCR gene therapy.

  View details for DOI 10.1002/eji.201948140

  View details for Web of Science ID 000478643000013

  View details for PubMedID 31054264

• Successive crystal structure snapshots suggest the basis for MHC class I peptide loading and editing by tapasin PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hafstrand, I., Sayitoglu, E., Apavaloaei, A., Josey, B., Sun, R., Han, X., Pellegrino, S., Ozkazanc, D., Potens, R., Janssen, L., Nilvebrant, J., Nygren, P., Sandalova, T., Springer, S., Georgoudaki, A., Duru, A., Achour, A. 2019; 116 (11): 5055–60

  Abstract

  MHC-I epitope presentation to CD8+ T cells is directly dependent on peptide loading and selection during antigen processing. However, the exact molecular bases underlying peptide selection and binding by MHC-I remain largely unknown. Within the peptide-loading complex, the peptide editor tapasin is key to the selection of MHC-I-bound peptides. Here, we have determined an ensemble of crystal structures of MHC-I in complex with the peptide exchange-associated dipeptide GL, as well as the tapasin-associated scoop loop, alone or in combination with candidate epitopes. These results combined with mutation analyses allow us to propose a molecular model underlying MHC-I peptide selection by tapasin. The N termini of bound peptides most probably bind first in the N-terminal and middle region of the MHC-I peptide binding cleft, upon which the peptide C termini are tested for their capacity to dislodge the tapasin scoop loop from the F pocket of the MHC-I cleft. Our results also indicate important differences in peptide selection between different MHC-I alleles.

  View details for DOI 10.1073/pnas.1807656116

  View details for Web of Science ID 000460911500051

  View details for PubMedID 30808808

  View details for PubMedCentralID PMC6421438

• Functional Assessment for Clinical Use of Serum-Free Adapted NK-92 Cells CANCERS Chrobok, M., Dahlberg, C. M., Sayitoglu, E., Beljanski, V., Nahi, H., Gilljam, M., Stellan, B., Sutlu, T., Duru, A., Alici, E. 2019; 11 (1)

  Abstract

  Natural killer (NK) cells stand out as promising candidates for cellular immunotherapy due to their capacity to kill malignant cells. However, the therapeutic use of NK cells is often dependent on cell expansion and activation with considerable amounts of serum and exogenous cytokines. We aimed to develop an expansion protocol for NK-92 cells in an effort to generate a cost-efficient, xeno-free, clinical grade manufactured master cell line for therapeutic applications. By making functional assays with NK-92 cells cultured under serum-free conditions (NK-92SF) and comparing to serum-supplemented NK-92 cells (NK-92S) we did not observe significant alterations in the viability, proliferation, receptor expression levels, or in perforin and granzyme levels. Interestingly, even though NK-92SF cells displayed decreased degranulation and cytotoxicity against tumor cells in vitro, the degranulation capacity was recovered after overnight incubation with 20% serum in the medium. Moreover, lentiviral vector-based genetic modification efficiency of NK-92SF cells was comparable with NK-92S cells. The application of similar strategies can be useful in reducing the costs of manufacturing cells for clinical use and can help us understand and implement strategies towards chemically defined expansion and genetic modification protocols.

  View details for DOI 10.3390/cancers11010069

  View details for Web of Science ID 000457233300033

  View details for PubMedID 30634595

  View details for PubMedCentralID PMC6356567