Cell line and use thereof

Abstract

The present invention provides a cell line derived from undifferentiated pleomorphic sarcoma that expresses PD-L1 and collagen and is deposited in the European Collection of Authenticated Cell Cultures under accession number 22113001. This cell line is particularly suitable for immunotherapeutic research, immune cell production and xenotransplantation and / or xenografting in animals.

Description

[0001] Cell line and use thereof

[0002] Field of Art

[0003] The present invention relates to an undifferentiated pleomorphic sarcoma cell line expressing PD-L1 and collagen.

[0004] Background Art

[0005] Undifferentiated pleomorphic sarcoma (UPS) is a type of cancerous diseases that develops typically in the soft tissues of the body. It is one of the most common aggressive sarcomas, while it is still poorly characterized. Treatment procedures include surgical removal of tumor tissue, radiotherapy, chemotherapy, and immunotherapy.

[0006] However, immunotherapy of UPS is not well explored due to its heterogeneity, antigenic variability, and lower tumor infiltration by cells of the immune system. Also, not too many UPS-derived cell lines have been prepared so far. Of a total of 844 sarcoma lines derived from patient-derived tumor tissues, only 43 are derived from UPS, and of these only 5 lines are publicly available.

[0007] Immunotherapy is a promising treatment procedure for UPS, which is one of the few types of soft tissue sarcoma that responds well to immunotherapy. Particularly good results were observed with immunotherapy based on inhibition of the PD-1 receptor / PD-U1 ligand interaction. The inhibitory receptor PD-1 is expressed by T cells of the immune system, and PD-U1 is the PD-1 ligand expressed by tumor cells. Binding of PD-1 to PD-U1 disrupts T cell function. Anti -PD-1 and anti-PD-Ul antibodies block this interaction and thus increase the immune response against tumor cells.

[0008] Recently, it has been found that another receptor expressed on T cells of the immune system, UAIR-1 (leukocyte-associated Ig-like receptor- 1), can be bound by collagen produced by some tumor cells or tumor-associated fibroblasts, which also reduces the antitumor function of T cells.

[0009] Currently, there is essentially no suitable cell line of undifferentiated pleomorphic sarcoma that would be suitable for testing and developing further immunotherapeutic procedures reflecting these latest findings.

[0010] Disclosure of the Invention

[0011] Object of the present invention is a new cell line of undifferentiated pleomorphic sarcoma, derived from a tumor sample obtained from a patient. This cell line is the first undifferentiated pleomorphic sarcoma cell line to express both PD-U1 (PD-1 receptor ligand) and collagen (UAIR-1 receptor ligand). The line was obtained from a tumor from a patient's thigh. The source tissue was collected before the patient was subjected to treatment.

[0012] The cell line has been deposited in The European Collection of Authenticated Cell Cultures (ECACC), UK Health Security Agency, Culture Collections, Porton Down, Salisbury, Wiltshire SP4 OJG, United Kingdom, under accession number 22113001, deposit date: 30 / 11 / 2022, depositor : Daniel Smrz, Department of Immunology, Charles University, Second Faculty of Medicine, V Uvalu 84, Prague 5, 150 06, Czech Republic). The inventors designated the line as JBT19.

[0013] The cell line designations JBT19 and ECACC 22113001 are used interchangeably in this text.

[0014] The cell line was phenotypically characterized by microscopy and flow cytometry. Analyses showed that the new cell line JBT19 expresses collagen, nestin, vimentin, aSMA, and Ki-67, and shows surface expression of PD-L1, FAP, CD95 (Fas), CD44, and CD47. However, the new cell line JBT19 does not show surface expression of PD-1, Tim-3, Gal-9, CD34, CD117, CD133, TRAIL, DR3, CD95L (FasL), CD30 and CD31. The new cell line JBT19 expresses on its surface the antigen presenting receptor MHC-I, but not MHC-II.

[0015] Simultaneous expression of PD-L1 and collagen is a unique feature of the new cell line JBT19. This simultaneous expression makes JBT19 cells very suitable for testing immunotherapeutics in vitro (or ex vivo), especially immunotherapeutics for immunotherapies based on adoptive transfer of cells of the immune system (adoptive cellular immunotherapy) or inhibition of immune checkpoint inhibitors (ICI) PD-1 and LAIR-1.

[0016] The PD-1 ligand, PD-L1, is one of the most targeted molecules in cancer immunotherapy. However, targeting this molecule is not always successful in solid tumors. This failure is often caused by multiple mechanisms, including co-expression of inhibitory molecules. One of the inhibitory molecules used by solid tumors is collagen. Collagen not only forms a physical barrier that protects the tumor from immune cell infiltration but is also a ligand for inhibitory receptors expressed on the surface of immune cells, especially for leukocyte-associated immunoglobulin receptor 1 (LAIR-1). LAIR- 1 -mediated signaling suppresses T cell activation and induces immunosuppression. Experiments showed that 2D and 3D cultured JBT19 cells express collagen, and when these cells are xenografted into immunodeficient athymic nude (nu / nu) mice, they form solid tumors with high collagen content. Because these tumors also express PD-L1, JBT19 cells represent a unique model for investigating the interplay of PD-1 and LAIR-1 in the resistance of solid tumors to immunotherapy. This uniqueness is particularly emphasized in light of recent findings showing that collagen promotes anti-PD-l / PD-Ll resistance in cancer through LAIR1 -dependent exhaustion of CD8+ T cells. Furthermore, JBT19 cells can be used for ex vivo production of immune cells reactive to JBT19 cells, in particular NK cells, CD8+ T cells and CD8+NKT-like cells, from allogeneic and autologous lymphocytes. These reactive cells of the immune system can be used to treat sarcomas or to research sarcoma treatments.

[0017] JBT19 cells can also be xenotransplanted / xenoimplanted (xenografted) into animals, preferably into mice, in particular for the purpose of studying UPS and studying possible UPS therapies.

[0018] Preliminary experiments also showed that 2D cultured JBT19 cells show sensitivity to selected active pharmaceutical substances (APIs), such as inhibitors of the signaling pathway of mTOR complexes (rapamycin, Torin 1). These findings show that JBT19 can be used in research and testing of APIs for chemotherapy or its combination with other therapeutic approaches, especially immunotherapy.

[0019] The inhibitory effect of the collagen / UAIR- 1 axis on immune cells determines that immunotherapy research, more specifically adoptive cell immunotherapy and ICIs immunotherapy research, or its combination with other therapeutic approaches, is the primary area of use for the JBT19 cells. Immune cell populations reactive to JBT19 cells can be generated ex vivo from both allogeneic and autologous lymphocytes. Experiments have shown ex vivo generation of JBT19-reactive NK cells, CD8+ T cells and CD8+NKT-like cells. Recently, CD8+NKT-like cells were found to exhibit dual toxicity and kill not only tumor cells but also suppressor cells derived from the myeloid lineage in an antigen-specific manner (Ei Z, Wu Y, Wang C, Zhang M (2019) Cancer immunology, immunotherapy : CII 68: 1303- 1315 doi: 10.1007 / s00262-019-02363-3). The data generated within the framework of the present invention showed that ex vivo generated lymphocytes containing JBT19-reactive immune cell populations were able to eradicate (eliminate) JBT19 cells in 2D cell culture. Thus, the JBT19 cells were immunogenic and sensitive to cytotoxicity of reactive lymphocytes and can be used in immunotherapeutic research as target cells or as cells for ex vivo generation of JBT19-reactive immune cells.

[0020] The reaction of the immune system against tumor cells can shape their phenotype, including changes in the surface expression of immunoregulatory molecules. JBT19 cells can strongly respond to the immune reaction against them. Specifically, the cytokines produced mainly by cytotoxic lymphocytes, interferon (IFN) gamma, was found not only to enhance the surface expression of the antigen- presenting receptor MHC-I but also to enhance the expression of PD-L1, thus presumably promoting anti-PD-l / PD-Ll -mediated resistance of JBT19 tumors. Moreover, the impact of IFN gamma on the expression of antigen-presenting receptor MHC-I is also elicited by other cytokines, tumor necrosis factor (TNF) alpha or IFN alpha. On the other hand, these two cytokines do not promote the expression of PD-L1. However, TNF alpha is still capable of further promotion of IFN gammamediated enhancement of PD-L1 expression.

[0021] Effective immunotherapy largely relies on the recognition of tumor cells via the surface expression of antigen-presenting receptor MHC-I on their surface. However, a subset of tumors also express on their surface the antigen presenting-receptor MHC-II, which is traditionally associated with professional antigen-presenting cells like dendritic cells (Axelrod ML, Cook RS, Johnson DB, Balko JM (2019) Clin Cancer Res 25:2392-2402 doi: 10.1158 / 1078-0432. CCR-18-3200). This receptor is necessary for antigen presentation to CD4+ T cells, whose role is increasingly investigated in tumor biology and immunotherapy. The expression of this receptor is increasingly found to be associated with favorable prognosis and response to immunotherapy. Although JBT19 cells are negative for the surface expression of MHC-II receptor, their treatment with IFN gamma induces its de novo expression on their surface, turning MHC-II-negative JBT19 cells into MHC-II+ JBT19 cells. In addition, although the other two cytokines, TNF alpha and IFN alpha, are not able to de novo induce the surface expression of MHC-II receptor, they can differentially impact the IFN gamma-induced de novo surface expression of MHC-II receptor - TNF alpha promotes this expression, IFN alpha does the opposite.

[0022] The impact of the IFN gamma-induced de novo surface expression of MHC-II receptor is sustainable for several days, even after removing this cytokine. A similar result is also true for the IFN gammainduced enhanced surface expression of the antigen-presenting receptor MHC-I. Importantly, the de novo expressed MHC-II receptor is functional as it can stimulate CD4+ T cells. Similar is determined for the expression of MHC-I receptor, whose IFN gamma-induced enhanced surface expression is associated with stronger stimulation of CD8+ T cells. The functionality of the expressed markers could be thus used as surrogate markers of their expression.

[0023] The sustainability of the cytokine -induced enhanced surface expression of PD-L1 in JBT19 cells is different. Unlike MHC-I or MHC-II receptors, the removal of the cytokine leads to a partial reversion of the enhancement in 2 days, showing the plasticity of the cells in the PD-L1 surface expression in response to external stimuli, such as IFN gamma. As such features of JBT19 cells can in vitro recapitulate the plasticity of PD-L1 expression in tumor samples, JBT19 can represent a novel model for studying or testing the mechanisms of this plasticity in vitro using either defined stimulating agents or biological samples obtained from patients. This model can also have significant clinical ramifications as the expression of PD-L1 in tumors does not necessarily, due to the plasticity of its expression, translate into the patient's response to the therapy based on PD-1 / PD-L1 checkpoint inhibitors (Shklovskaya E, Rizos H (2020) International journal of molecular sciences 21 doi: 10.3390 / ijms21197139, Kuo MH et al. (2021) Stem Cells 39: 1298-1309 doi: 10.1002 / stem.3429).

[0024] Apart from the above-noted surface molecules, IFN gamma and TNF alpha also enhance the surface expression of CD47 and CD44, and IFN gamma enhances the expression of Fas (CD95). CD47 is a “don’t eat me” receptor, immunotherapeutically targeted by blocking antibodies, such as Magrolimab. Fas (CD95) is a receptor for Fas ligand (FasL, CD95L), and its stimulation induces cell apoptosis. On the one hand, these two cytokines can thus increase the resistance of JBT19 cells to their CD47- mediated phagocytosis by immune cells (Ye ZH et al. (2021) Translational oncology 14: 101162 doi: 10.1016 / j.tranon.2021.101162), but, on the other hand, sensitize them to CD95 -mediated apoptosis (Liu H et al. (2023) Cancer Cell 41:693-710 e698 doi: 10.1016 / j.ccell.2023.03.004). CD44 is a stern cell marker whose expression in tumor cells is associated with increased resistance to therapy (Xu H et al. (2020) Experimental hematology & oncology 9:36 doi: 10.1186 / s40164-020-00192-0). The CD44 is a marker of increased cancer cell sternness (Yan Y et al. (2015) Stem cells translational medicine 4: 1033-1043 doi: 10.5966 / sctm.2015-0048).

[0025] The reaction of the immune system against tumor cells can also elicit the immune response of the tumor cells themselves through the production of biologically active products, such as cytokines. JBT19 cells were found to produce the cytokine IL-8 in response to stimulation with TNF alpha. IL8 is known not only to largely modulate the immune responses, such as recruiting pro-tumorigenic immune cells into the tumor microenvironment (Fousek K, Hom LA, Palena C (2021) Pharmacology & therapeutics 219: 107692 doi: 10.1016 / j.pharmthera.2020. 107692), but it can also increase proliferation of tumor cells and inhibit their apoptosis (programmed cell death) (Guo Y, Zang Y, Lv L, Cai F, Qian T, Zhang G, Feng Q (2017) Molecular medicine reports 16:9035-9042 doi: 10.3892 / mmr.2017.7747). With respect to the PD-1 / PD-L1 -mediated tumor resistance, neutralization of IL-8 was also recently found to potentiate anti-PD-1 immune checkpoint blockade efficacy for glioma (Liu H et al. (2023) Cancer Cell 41:693-710 e698 doi: 10.1016 / j.ccell.2023.03.004), thus indicating that IL-8 could be considered as a soluble target for combined immunotherapy. Therefore, JBT19 cells could be used as a model to study IL-8 as a target for immunotherapy.

[0026] Collectively, JBT19 cells were found to be responsive to the biologically active components of the immune system, and this responsiveness was altering their phenotype and activity associated with multiple mechanisms of either tumor resistance or sensitivity to the immune system and therapeutic interventions. As such, JBT19 cells can be used in immunotherapeutic and other oncologic research as a model cell for in vitro and in vivo investigation of the mechanisms of tumor resistance or sensitivity towards the immune system and therapeutic interventions.

[0027] A further aspect of the invention is thus an in vitro method of detecting inflammatory cytokines in a biological sample, comprising a step of contacting JBT19 cells with the biological sample and a subsequent step of detecting expression of MHC-II and / or IL-8, and / or detecting the extent of MHC-I, PD-L1, CD47, Fas (CD95), and / or CD44 expression in the JBT19 cells. Expression of MHC-II and / or IL-8, and / or higher extent of MHC-I, PD-L1, CD47, Fas (CD95), and / or CD44 expression show the presence of inflammatory cytokines, and thus the presence of adaptive immune response in the biological sample or in the patient from whom the biological sample was taken. The term “higher extent” relates to an extent of expression higher than in a previous measurement for the same patient, or an extent of expression higher than in healthy human. As an alternative to detecting the changes in the expression or changes in the extent of the expression of the aforementioned markers, the functionality of these markers could be used as a surrogate marker of their expression. The presence of an adaptive immune response indicates the success of the treatment given to the patient, which should be continued, while its absence indicates that a different treatment should be administered to the patient. A biological sample may be, for example, serum, exudate, disintegrated tumor tissue.

[0028] The new cell line was prepared using a procedure for the preparation of tumor cell lines from tumor tissue, in which the tumor tissue is disintegrated to form a cell suspension, to which an anti-TNF-a antibody is added, and the cell suspension is further cultured under adherent conditions. This procedure forms another aspect of the invention.

[0029] The addition of the anti-TNF-a antibody solves problems occurring when establishing new cell lines, namely the problems caused by the presence of tumor infdtrating lymphocytes. Tumor-infiltrating lymphocytes are a mixture of cells of the immune system that enter the tumor and that are usually enriched with cell populations, in particular so-called cytotoxic lymphocytes, which are able to specifically recognize and eliminate tumor cells. The presence of these cells can then significantly reduce the success rate of establishing cell lines. Addition of anti-TNF-a antibody minimizes the effect of tumor infiltrating lymphocytes on cell line establishment.

[0030] In case of passage or exchange of medium, it is highly recommended that the added medium also contains anti-TNF-a antibody.

[0031] Brief Description of Drawings Figure la on the left shows the growth curve based on which the doubling time of the number of JBT19 cells in the adherent cell culture was determined to be 73.8 hours, Figure la on the right shows the appearance of the adherent culture under the microscope. Figure lb shows a microscope image of a 14-day-old spheroid formed by 14-day cultivation of JBT19 cells in 3D cell culture. Figure 1c shows fluorescent antibody-labeled PD-L1 molecules (left) and collagen (middle) in adherent JBT19 cells and collagen in a 14-day spheroid of JBT19 cells (right) under the microscope.

[0032] Figure 2a shows the flow cytometer data processing procedure. Figure 2b and 2c show a histogram of negative (molecule not present; Tim-3 and Fas-L) and positive (molecule present; Ki-67, PD-L1, Collagen, aSMA, nestin and vimentin) intracellular fluorescent labeling intensities (PE, FITC or AF488) of the indicated molecules measured by flow cytometer and processed as shown in Fig. 2a. Ctrl indicates a sample without added antibody. Figure 2d shows a histogram of positive intensities (molecule present) of surface fluorescent labeling (PE, APC) of the indicated molecules measured by flow cytometer and processed as shown in Fig. 2a. Ctrl indicates a sample without added antibody. Figure 2e shows a histogram of negative intensities (molecule not present) of surface fluorescent labeling (PE-Cy7, FITC, APC, PE) of the indicated molecules measured by flow cytometer and processed as shown in Fig. 2a. Ctrl indicates a sample without added antibody. Figure 2f shows on the left a histogram of positive intensity (molecule present) and on the right a histogram of negative intensity (molecule not present) of the surface fluorescence labeling (PE, APC) of the indicated molecules measured by flow cytometer and processed as shown in Fig. 2a. Ctrl indicates a sample without added antibody.

[0033] Figure 3a shows STR (Short Tandem Repeats) analysis of JBT19 cells. Figure 3b shows the HLA genotype specification of classical and non-classical loci of JBT19 cells and cells from the patient's peripheral blood.

[0034] Figure 4 shows the karyotype analysis of JBT19 cells.

[0035] Figure 5a shows a developed solid JBT19 tumor (black arrow) in immunodeficient athymic nude (nu / nu) mice after xenoimplantation of JBT19 cells. Figure 5b shows solid JBT19 tumor growth in 6 immunodeficient athymic nude (nu / nu) mice after xenoimplantation of JBT19 cells. Figure 5c shows collagen in a JBT19 tumor section from nude mice visualized by two-photon confocal microscopy (SHG).

[0036] Figure 6 shows the results of cell populations of allogeneic (HD) and autologous (Pac) lymphocytes expanded using the K-562 cell line (K-562-primed) or the JBT19 cell line (JBT19-primed). The data show the rate of cell culture expansion, viability and representation of individual cell populations based on data processing as shown in Fig. 7a.

[0037] Figure 7a shows the flow cytometer data processing procedure. Figure 7b shows the results of measured intracellular labeling data of the pro-inflammatory cytokines TNF-alpha and IFN-gamma in allogeneic (HD) and autologous (Pac) lymphocytes expanded using the K-562 cell line (K-562- primed). Expanded lymphocytes were stimulated with K-562 (K-562-stim) or JBT19 (JBT19 stim) and the percentage of reactive (specific) lymphocytes were detected as the percentage of cells producing the pro-inflammatory cytokines TNF-alpha and IFN-gamma, which were detected by intracellular labeling using fluorescently labeled antibodies.

[0038] Figure 8a shows the results of measuring intracellular labeling data of the pro-inflammatory cytokines TNF-alpha and IFN-gamma in allogeneic (HD) and autologous (Pac) lymphocytes expanded using the JBT19 cell line (JBT19-primed). Expanded lymphocytes were stimulated with K-562 (K-562-stim) or JBT19 (JBT19-stim), and the percentage of reactive (specific) lymphocytes was detected as the percentage of cells producing the pro-inflammatory cytokines TNF-alpha and IFN-gamma, which were detected by intracellular labeling using fluorescent labeled antibodies. Figure 8b shows the cytotoxic activity of the expanded cells in Fig. 7b and 8a using transfected fluorescent (TurboGFP) JBT19 cells (persistently producing the fluorescent protein TurboGFP). TurboGFP-JBT19 were cocultured with expanded lymphocytes at a ratio of 1:5. The amount of labeled JBT19 cells in the coculture was determined as a measure of fluorescence in the coculture. Cytotoxic activity was determined as a measure of the decrease in fluorescence in the coculture.

[0039] Figure 9 shows the impact of 2-day treatment of JBT19 cells with IFN gamma (IFNy, 200 ng / ml), TNF alpha (TNFa, 20 ng / ml), IFN alpha (IFNa, 200 ng / ml), or their combinations (IFNy+TNFa, IFNy+INFa) on the surface expression of MHC-I receptor, PD-L1, or MHC-II receptor. The panels in Figure 9 show histograms of the expression intensities, and the panels in Figure 9 - cont. show the corresponding graphs with statistical evaluations of the expression intensities (MFI, one-way ANOVA).

[0040] Figure 10 shows the impact of the treatment of JBT19 cells with IFN gamma (IFNy, 200 ng / ml) on the surface expression of MHC-II or MHC-I receptor and the sustainability of this impact. The treatment was performed either for 4 days (4d) or for 2 days, followed by an extensive rinse with medium and 2- day cell culturing to evaluate the reversion of the IFN gamma-induced phenotype (2d+2drev). The upper panels show histograms of the expression intensities, and the bottom panels show the corresponding graphs with the expression intensities (MFI). Figure 11 shows the impact of the treatment of JBT19 cells with IFN gamma (IFNy, 200 ng / ml) on the surface expression of PD-L1. The treatment was performed either for 4 days (4d) or for 2 days, followed by an extensive rinse with medium and 2-day cell culturing to evaluate the reversion of the IFN gamma-induced phenotype (2d+2drev). The upper panels show histograms of the expression intensities, and the bottom panels show the corresponding graph with the expression intensities (MFI).

[0041] Figure 12 shows the impact of the treatment of JBT19 cells with IFN gamma (IFNy, 200 ng / ml) or TNF alpha (TNFa, 20 ng / ml) on the surface expression of CD47, Fas (CD95), FAP, or CD44. The treatment was performed for 2 days (CD47, Fas (CD95), FAP) or 8 days (CD44). The panels in Figure 12 show histograms of the expression intensities of CD47, Fas (CD95), or FAP. The graphs in Figure 12 - cont. show the expression intensities (MFI) of CD47, Fas (CD95), FAP, or CD44.

[0042] Figure 13 shows the results of measured intracellular labeling of the cytokine IL-8 in JBT19 cells treated for 7 days with TNF alpha (TNFa, 20 ng / ml). The production of IL-8 in the TNF alpha-treated (7-d TNFa treatment) or non-treated (No treatment) JBT19 cells was detected as the percentage of cells producing IL-8 cytokine, which was detected by intracellular labeling using fluorescently labeled antibody.

[0043] Figure 14 shows the results of measured intracellular labeling data of the pro-inflammatory cytokines TNF-alpha and IFN-gamma in allogeneic lymphocytes expanded using 2-day IFN gamma-treated JBT19 cells (IFNy, 200 ng / ml). Expanded lymphocytes were then stimulated with JBT19 cells not treated (JBT19 stim) or 2-day treated with IFN gamma (200 ng / ml, IFNy-treated JBT19 stim), and the percentage of reactive (specific) lymphocytes, CD4+ or CD8+ T cells, were detected as the percentage of cells producing the pro-inflammatory cytokines TNF-alpha and IFN-gamma, which were detected by intracellular labeling using fluorescently labeled antibodies.

[0044] Examples

[0045] Cell line generation

[0046] The source tissue donor was a 65 -year-old patient in August 2019. During a planned operation, a tissue mass (18 x 14 x 5 cm) with the tumor was excised from the patient's thigh. The tumor mass was subsequently divided for histological examination and further processing. Histological examination of the excised tissue confirmed the presence of a solid tumor mass (8 x 5 x 8 cm) diagnosed as grade 3 UPS. Six months later, in January 2021, the patient was diagnosed with a metastatic form of UPS. The patient provided pre-signed written informed consent for the use of a portion of the excised tissue for future research. The research was approved according to the ethical standards of the institutional research committee - the Ethics Committee of the Motol Faculty Hospital in Prague and was conducted in accordance with the Declaration of Helsinki from 1964 and its later amendments or comparable ethical standards.

[0047] Establishment of the cell line

[0048] The establishment of cell lines from primary or metastatic tumor tissue is achieved either by direct in vitro cultivation of processed tumor tissue or by a combination of xenografting of processed tumor tissue and subsequent in vitro cultivation of processed xenografted tumors. The success rate of establishing new lines is usually not high and depends on the type of tumor, the stage of the tumor disease and the establishment technique, which includes methods of processing the tumor tissue and subsequent cultivation.

[0049] A major problem in establishing new cell lines is the presence of tumor-infiltrating lymphocytes. Tumor-infdtrating lymphocytes are a mixture of cells of the immune system that enter the tumor and which are usually enriched with cell populations, in particular so-called cytotoxic lymphocytes, which are able to specifically recognize and eliminate tumor cells. The presence of these cells can then significantly reduce the success rate of establishing cell lines. When establishing the new undifferentiated pleomorphic sarcoma (UPS) cell line, an immunosuppressive recombinant human monoclonal antibody (Adalimumab) directed against the pro-inflammatory cytokine TNF-a was therefore used, with the aim of minimizing the influence of tumor infiltrating lymphocytes on the establishment of the cell line.

[0050] The tumor tissue collected from the patient was placed in LM medium [RPMI 1640 (Thermo Scientific, Waltham, MA, USA) containing 5% human plasma serum (One Eambda, Canoga Park, CA, USA), 100 U / ml penicillin-streptomycin, 2 mM GlutaMax, 1 mM sodium pyruvate and a mixture of non-essential amino acids (Thermo Scientific)] and transported in a sealed container to the laboratory, where it was further processed under sterile conditions. The tumor tissue was first rinsed several times with PBS and placed in a Petri dish, where it was mechanically disintegrated using surgical scissors. Such disintegrated tissue was further processed as described in our earlier publications (Strizova Z, Taborska P, Stakheev D, Partiova S, Havlova K, Vesely S, Bartunkova J, Smrz D (2019) Urologic oncology, 37, 503-509, doi: 10.1016 / j.urolonc.2019.03.014; Strizova Z, Bartunkova J, Smrz D (2019) Cancer immunology, immunotherapy : CII, 68, 1831-1838, doi: 10.1007 / s00262-019-02359-z). The use of this technique allows for a high degree of tissue loosening and transfer of tumor cells into a cell suspension, thus simultaneously increasing the probability of survival of tumor cells in cell culture. However, this technique is initially used to expand infiltrating lymphocytes using high concentrations of lymphocyte growth factors, such as IL-2, for cancer cellular immunotherapy (Rosenberg SA, Restifo NP (2015) Science 348:62-68 doi: 10. 1126 / science.aaa4967). It is the high degree of tissue loosening with this technique that can significantly increase the risk that free suspended tumor infiltrating lymphocytes attack and kill tumor cells, thereby making it impossible forthem to "attach" in 2D culture, especially when tumor disintegration disrupts the tumor microenvironment, which ensures protection of tumor cells from the immune system. For this reason, an anti-TNF-a antibody (Adalimumab) was added to the tumor suspension. The cell suspension in the presence of the anti-TNF-a antibody was added to a 6-well flat bottom plate (TPP, Trasadingen, Switzerland) and supplemented with LM medium. The plate with the processed material was then cultured (37 °C, 5% CO2). The presence of the immunosuppressive antibody in the culture with its regular replenishment allowed to extend the time (to 8 days) for tumor cells to "attach" (adhere) to the surface of the culture well. During this time, the medium was halfdepleted twice and supplemented with fresh LM medium with the anti-TNF-a antibody (Adalimumab). After 8 days of cell culture, the non-adherent fraction was removed from the plate and the adherent cells were rinsed with PBS and harvested after treatment with trypsin / EDTA solution (Thermo Scientific). Cell suspensions were pelleted by centrifugation and passaged into T75 tissue flasks in LM medium (TPP). Although passage of cells washes away the non-adherent fraction of the cell culture, which also contains tumor-infiltrating lymphocytes, and subsequent rinsing with PBS removes majority of the immune cells, many immune cells, including cytotoxic lymphocytes, can, like tumor cells, be adhered and after the passage then continue to remain in the cell culture. These are mainly activated lymphocytes, which can show an increased ability to adhere. Therefore, subsequent cultivations after this and other passages were carried out in the presence of anti-TNF-a antibody, even at lower concentrations (Table 1).

[0051] Table 1:

[0052] The cells were cultured for over 30 months and underwent 30 passages during this cell culture period. Cells were found to be well cryopreservable in LM medium with 10% DMSO and well reconstitutable back into culture after 12 months of storage in liquid nitrogen. Reconstitution of the cell culture was also feasible after several cycles of cryopreservation.

[0053] In the 2D cell culture format, the growth rate of cells with a 3 -day duplication period is shown in Fig. la, left panel. Cells with expanded fibroblast-like shapes tend to line up in parallel in the cell culture (Fig. lb, right panel). JBT19 cells in 2D culture also showed a tendency to clump and layer when overgrown. JBT19 cells were found to form growing spheroids in low-adherence dishes (not shown). The ability of JBT19 cells to form spheroids up to 300 micrometers in size was subsequently confirmed by microscopic observation (Fig. lb and 1c).

[0054] Phenotypic characterization

[0055] For cell surface or intracellular expression profiling, adherent JBT19 cells were harvested by trypsinization and washed with LM medium and PBS with 2 mM EDTA (PBSE). Cells were stained as described in Taborska P, Stakheev D, Svobodova H, Strizova Z, Bartunkova J, Smrz D (2020) Cancers 12 doi: 10.3390 / cancersl2123766, with the following fluorescently labeled antibodies: CD95(Fas)-APC (clone DX2), CD178(FasL)-PE (clone NOK-1), CD31-PC7 (clone WM59 ), CD47- APC (clone CC2C6), CD263(TRAIL)-APC (clone RIK-2), CD279(PD-1)-APC (clone EH12.2H7), CD30-APC (clone BY88), CD366(Tim- 3)-PE (clone F38-2E2), Galektin-9-PE (clone 9M1-3), CD274(PD-L1)-APC (clone MIH3), HLA-ABC(MHC-I)-PE (clone W6 / 32), HLA-DP, DQ, DR(MHC-II)-APC (clone Tti39) (BioLegend, San Diego, CA), fibroblast activation protein a(FAP)- PE (clone 427819), DR3-PE (clone 59204) (R&D Systems, Minneapolis, MN, USA), CD117(c-Kit)- PE (clone YB5.B8) (Becton Dickinson, Franklin Lakes, NJ, USA), CD34-FITC (clone AC136), CD133 / 1-APC (clone AC133) (Miltenyi Biotec, Gladbach, Germany), CD44-PE (clone MEM-263) (Exbio, Prague, Czech Republic). The following dye was used for intracellular staining: COL1A1 XP- AlexaFluor647 (clone E8F4L) (Cell Signaling, Danvers, MA, USA), a-smooth muscle actin (aSMA)- APC (clone 1A4), vimentin-FITC (clone V9), nestin-AF488 (clone 10C2). Stained cells were analyzed by flow cytometry (FACSAria II or FACSFortesa, Becton Dickinson, Heidelberg, Germany) and FlowJo software (Tree Star, Ashland, OR) was used for data analysis.

[0056] Phenotypic characterization was performed using microscopy and flow cytometry. Analyses showed that JBT19 cells expressed PD-L1 (Fig. 1c, left panel) and collagen (Fig. 1c, middle and right panels). Extended / confirmatory phenotypic analysis confirmed PD-L1 and collagen expression (Fig. 2c, left panel). The analysis further showed the expression of aSMA, nestin and vimentin (Fig. 2c, right panel) and the proliferation marker Ki-67 (Fig. 2b). Analysis also showed surface expression of FAP, CD95 (Fas), CD44, CD47 and PD-L1 (Fig. 2d). On the other hand, JBT19 cells were found to be negative for the surface expression ofPD-1, Tim-3, Gal-9, CD34, CD117, CD133, TRAIL, DR3, CD95L (FasL), CD30 and CD31 (Fig. 2e). The cells were also found to express antigen presenting receptor MHC-I but not MHC-II molecules (Fig. 2f).

[0057] Cell line validation, HLA-genotyping and karyotype analyses

[0058] Cell line validation was performed by genetic testing. Genomic DNA from cells was isolated using the MagCore® Genomic DNA Whole Blood Kit (RBC Bioscience, Taipei, Taiwan) using paramagnetic beads in the presence of chaotropic salts according to the manufacturer's instructions. Cell line validation was determined by short tandem repeat (STRs) polymorphism genotyping by fragment analysis using the PowerPlex® 16 HS System (Promega, Madison, WI, USA). HLA-genotyping was performed by Next Generation Sequencing using the AlloSeq Txl7kit (CareDx, Brisbane, CA, USA). Obtained sequences were analyzed using AlloSeqAssignv.1.0.3.1331, IMGT / HLA3.45. 1. 1 database (accessed 2022-07-12). Allelic balance was analyzed according to standardized procedures.

[0059] Karyotype - Adherent JBT19 cells in cell culture were treated with demecolcine for 24 hours before harvesting. Harvesting and preparation of slides were performed according to standard cytogenetic procedures. Harvested cells were stored at -20°C in methanol-glacial acetic acid (ratio 3: 1). For cytogenomic analyses, cell suspensions were dropped onto microscope slides and air-dried. Multicolor fluorescence in situ hybridization (mFISH) was used to characterize the chromosomes in detail. Analyses were performed using the commercially available 24 probe XCyte mFISH Kit (MetaSystems, AltluBheim, Germany). All available metaphases were scanned using a Metafer Axiolmager Z2 - an automatic metaphase finder and an Axiolmager Z1 fluorescence microscope (Carl Zeiss, Jena, Germany) and further analyzed using the Isis computerized analysis system (MetaSystems). Findings were described according to ISCN 2020 (McGowan-Jordan J, Hastings RJ, Moore S (2020) ISCN 2020: An International System for Human Cytogenomic Nomenclature (2020). Karger, Basel, doi: 10. 1159 / isbn.978-3-318-06867-2). The obtained JBT19 cells were validated by 15 STR loci located on 13 chromosomes and 1 sexspecific locus. The data showed that for some loci it is not possible to say with certainty whether it is a real allele or an artifact of the PCR reaction, the so-called stutter peak. The stutter product is usually about 5 to 15% of the area of the real allele of a given locus. Peak imbalance was observed in heterozygotes. This is a common phenomenon in the amplification of cell lines. These alleles represent in the locus D3S1358 10 %, D21S11 40 %, D18S51 40 %, Penta E 55 %, D5S818 80 %, D7S820 57 %, vWA 34 % and D8S1179 43 % of the real allele area. Loss of heterozygosity occurred at the THO1 (1 lpl5.5), D13S317 (13q22-q31), Penta D (21q) and TPOX (2p24-2pter) loci (Fig. 3a). The HLA genotype of classical and non-classical loci is specified in Fig. 3b. No allelic imbalance was detected. Next, we investigated the HLA genotype of the patient's peripheral blood cells. Analysis showed that the HLA genotype of the patient's peripheral blood cells was identical to the HLA genotype of JBT19 (Fig. 3b), confirming the origin of the JBT19 cells from the patient.

[0060] UPS is highly heterogeneous with numerous defects in the karyotype of transformed cells (Steele CD et al. (2019) Cancer Cell 35:441-456 e448 doi: 10.1016 / j.ccell.2019.02.002). Therefore, we further investigated the karyotype of the obtained JBT19 cells. Using mFISH, we revealed that JBT19 cells are a near tetrapioid cell line (92±) with many numerical and structural chromosome changes (Fig. 4). We found aberrated XY sex chromosomes and numerous numerical and structural aberrations in all 16 analyzed metaphases. The line has a complex karyotype (>3 changes, of which at least two changes must be structural or not only numerical). The changes concern (practically) all chromosomes. These data indicated that JBT19 cells are indeed genetically highly transformed cells.

[0061] Description of JBT19 cell karyotype:

[0062] 98~109<4n>,XX,+del(X)(?),+der(X)t(X;?)(p?;?),der(Y)t(Y;21)(pl l.2;?)t(6;21)(?;?)x2,+l, der(l)del(l)(p?)t(l;l l)(q?;q?)x2,+del(l)(?)x2,+del(l)(?),+del(2)(q?12)x2,der(3)del(3)(p?)del(3)(q?)x2 ,der(3)t(3;17)(q?21;qll.2)x2,+der(3)t(3;5)(p?12;?)t(3;17)(q?21;ql l.2), der(4)del(4)(p?14)del(4)(q?13)x2,+der(4)t(4;13)(p?16;?)del(4)(q?)x2,-6,t(6;10)(pl2;pl2), +7,der(7)t(7; 15)(p?21 ;?)x2,+der(7)t(7; 15)(p?21 ;?)del(7)(q?),+der(7)del(7)(p? 12)t(7; 17)(q? 11.2;?)t(6 ; 1 7)(?;?),+8,+8,del(8)(p?)x2,+der(8)t(4;8)(?;p?11.2)x2,+9,+9,del(9)(?),der(9)t(9;19)(p?;?),-l l,- l l,der(l l)t(ll;19)(pl5;?)x2,der(12)(?),-13,der(13;15)(qlO;qlO),-14,-14, der(14)t(7;14)(?;p?13),del(15)(q?)x2,+der(15)t(9;15)(q?;p?12),+der(15)t(10;15)(q?;p?12), +16,der(16)t(l l;16)(?;q?22),der(16)t(16;?)(pl l.2;?)t(l l;16)(?;q?22),der(16)t(16;17)(pl l.2;?)t(l l;16)( ?;q?22),-17,-17,del(17)(q?), del(18)(q?),der(19)del(19)(?)t(3;19)(?;?)x2,der(19)t(19;21)(?;q?)x2,+der(19)t(12;19)(q?;?)t(3;19)x2,de r(20)t( 17;20)(?;?)x2,ider(20)(q? 10)ins(20; 16)(q? 12,?),der(20)t(5 ;20)(?;p?)ins(20; 16)(q? 12,?),- 21 ,der(21 )t(6;21 )(? ;q?)x2,+22,ider(22)(q 10)t(3 ;22)(?;q 11 ,2?)x2 [cp 16] In vivo murine xenograft model

[0063] To verify whether JBT19 cells have the ability to form solid tumors, JBT19 cells were xenografted into immunodeficient athymic nude (nu / nu) mice.

[0064] Six athymic nude mice (nu / nu) (AnLab, Prague, Czech Republic) were used for in vivo studies. Mice were inoculated with JBT19 cells (1 x 106SC) and then bred for the indicated times. We found that xenografted JBT19 cells could produce solid tumors (Fig. 5a) reaching an area of up to 40 mm2in 24 days (Fig. 5b). Importantly, the tumors still expressed collagen (Fig. 5c). These data indicated that JBT19 cells can be suitably used for in vivo animal studies.

[0065] Preparation of autologous and allogeneic JBT19-reactive T cells and NK cells

[0066] The starting material for the preparation of autologous JBT19-reactive lymphocytes was non-clotted peripheral blood collected during a planned medical examination of a patient. The source material for the preparation of allogeneic JBT19-reactive lymphocytes was from the buffy coat of 4 healthy donors (2 women, 30 and 50 years old, and 2 men, 34 and 39 years old) obtained from the Institute of Hematology and Blood Transfusion in Prague. Peripheral blood mononuclear cells (PBMC) were isolated and cryopreserved as previously described (Taborska P, Bartunkova J, Smrz D (2018) J Immunol Methods 458:63-73 doi: 10. 1016 / j.jim.2018.04.005; Taborska P, Stakheev D, Svobodova H, Strizova Z, Bartunkova J, Smrz D (2020) Cancers 12 doi: 10.3390 / cancersl2123766). The patient and the healthy volunteers provided signed written informed consent for the use of biological material for future research. The research was approved according to the ethical standards of the institutional research committee - the Ethics Committee of the Motol Faculty Hospital in Prague and was conducted in accordance with the Declaration of Helsinki from 1964 and its later amendments or comparable ethical standards. To prepare JBT19-reactive lymphocytes, cryopreserved PBMCs were reconstituted overnight (2.5 x 106cells / ml) in LM / KM medium (1: 1 ratio) with IL-2 (500 lU / ml; PeproTech, Rocky Hill, NJ, USA). The composition of KM medium was: RPMI 1640 medium (Thermo Scientific) with 10% fetal bovine serum (HyClone, GE Healthcare Life Sciences, South Logan, UT, USA), 100 U / ml penicillin-streptomycin, 2 mM GlutaMax (Thermo Scientific).

[0067] The reconstituted cells were then stimulated (day 0) with freshly inactivated JBT19 cells by gamma (96 Gy) and UV (312 nm, 2.55 J / cm2) irradiation performed as previously described (Taborska P, Lastovicka J, Stakheev D, Strizova Z, Bartunkova J, Smrz D (2021) Immunity, inflammation and disease 9: 1452-1467 doi: 10.1002 / iid3.496; Taborska P, Stakheev D, Svobodova H, Strizova Z, Bartunkova J, Smrz D (2020) Cancers 12 doi: 10.3390 / cancersl2123766). The JBT19 : PBMC ratio was 1 : 4, and the stimulation and subsequent cell culture was performed in LM / KM medium (1 : 1 ratio) supplemented with IL-2 (500 lU / ml). Every second and third day of cell culture, cells were supplemented with an equal volume of fresh LM / KM medium (1: 1 ratio) supplemented with IL-2 (500 lU / ml). On day 7 of culture, cultured cells were counted and supplemented with freshly inactivated JBT19 cells at a ratio of 1:4 (JBT19:PBMC) and IL-2 (500 lU / ml). The following second and third day of cell culture, the cells were supplemented with an equal volume of LM / KM medium (1: 1 ratio) supplemented with IL-2 (500 lU / ml). On day 14 of cell culture, cells were analyzed. K-562 cell line (CCL-243; ATCC, Manassas, VA, USA) was used as a control instead of JBT19 cells during the procedures. K-562 cells were maintained in KM medium.

[0068] We first investigated whether JBT19 cells could be attacked by immune cells in vitro. We used allogeneic PBMCs from healthy donors and autologous PBMCs from the patient as the source material of immune cells. To prepare immune cells attacking cancer cells, we expanded PBMCs in cell culture using the K-562 cell line in combination with IL-2. The K-562 cell line are blasts obtained from a patient with chronic myeloid leukemia (CML). These cells lack HLA-antigen expression and are used for ex vivo expansion and stimulation of NK cells. Using inactivated K-562 cells and IL-2, we stimulated and expanded the cultured PBMCs ex vivo. Flow cytometry analysis showed that the expanded cell cultures contained NK cells, CD4+ and CD8+ T cells, and CD8+ NKT-like cells (CD56+CD8+CD3+ cells (Romero-Olmedo AJ et al. (2021) Eur J Immunol 51:672-681 doi: 10.1002 / eji.202048941)) (Fig. 6). We found that the expanded cells contained populations ofNK, CD8+ T, and CD8+ NKT-like cells that were reactive to either K-562 or JBT19 cells. Reactivity was determined by intracellular TNF-alpha and IFN-gamma production in stimulated cells (Figs. 7 and 8) No significant reactivity to K-562 or JBT19 cells was found in expanded CD4+ T cells (Figs. 7 and 8).

[0069] Analyses of autologous and allogeneic JBT19-reactive T cells, NK cells, and NKT-like cells

[0070] Cells were analyzed with minor modifications using procedures described previously (Taborska P, Stakheev D, Svobodova H, Strizova Z, Bartunkova J, Smrz D (2020) Cancers 12 doi: 10.3390 / cancersl2123766). Lymphocytes activated (primed) with K-562 or JBT19 cells were cultured for 14 days, harvested, pelleted by centrifugation and resuspended in LM / KM medium (ratio 1: 1) with IL-2 (250 lU / ml). The cells were then stimulated with K-562 or JBT19 cells at a ratio of 1:5 (stimulant: lymphocytes). After 1 h of stimulation, cells were supplemented with Brefeldin A (BioLegend). Cells were stimulated for 5 h and then stained with Vivid Aqua fixable cell viability dye (Thermo Scientific, Waltham, MA, USA), fixed, permeabilized and stained with CD3-PerCP-Cy5.5, CD4-PE-Cy7 (eBiosciences, San Diego, CA, USA), CD8-Alexa Fluor 700 and CD56-FITC (Exbio) and TNF-alpha-APC and IFN-gamma-PE (Becton Dickinson). Stained cells were analyzed by flow cytometry as described above. Analysis of the cytotoxic impact of JBT19-reactive T cells, NK cells, and NKT-like cells on

[0071] JBT19 cells

[0072] JBT19 cells were transfected with MISSION® pLKO.l-puro-CMV-TurboGFP transduction particles (Sigma- Aldrich, St. Louis, MO, SHC003V) to generate JBT19 cells with sustained expression of the fluorescent protein TurboGFP (TurboGFP-JBT19). Transduction and antibiotic selection of transfected cells (0.5 to 1.0 pg / ml puromycin, Sigma-Aldrich) were performed as previously described (Stakheev D, Taborska P, Strizova Z, Podrazil M, Bartunkova J, Smrz D (2019) Scientific reports 9:4761 doi: 10. 1038 / s41598-019-41182-5). Cytotoxicity assay was performed by co-culturing adherent TurboGFP-JBT19 cells with 14-day-old cultured lymphocytes activated (primed) by K562 or JBT19 cells using procedures described previously. 0. 1 x 106TurboGFP-JBT19 cells were seeded in a 48-well flat-bottom plate (Nalgene) and cultured for 2 days. The supernatant was removed and 0.5 x 106lymphocytes in 1 ml of LM medium with IL-2 (250 lU / ml) were added. After lymphocyte sedimentation for 10 min, mean fluorescence intensity (MFI) of TurboGFP-JBT19 cells in the wells (day 0) was determined by fluorescence microscopy and image analysis as detailed in Stakheev D, Taborska P, Strizova Z, Podrazil M, Bartunkova J, Smrz D (2019) Scientific reports 9:4761 doi: 10.1038 / s41598-019-41182-5. Cells were then co-cultured for 6 days. On days 1, 4, and 6 of cell culture, MFI of TurboGFP -JBT 19 cells in the wells was determined as on day 0.

[0073] In the next step, we investigated whether replacing K-562 cells with JBT 19 cells for lymphocyte priming could also lead to the expansion of JBT19- and / or K-562-reactive lymphocytes. We found that using JBT 19 cells for lymphocyte priming also resulted in cell culture expansion (Fig. 6). We confirmed that the expanded cell cultures contained NK cells, CD4+ and CD8+ T cells, and CD8+ NKT-like cells (Fig. 6). Compared to expanded lymphocytes activated (primed) by K-562 cells (Fig. 7a), the cell cultures contained populations of NK, CD8+ T, and CD8+ NKT-like cells that were reactive with either K-562 or JBT 19 cells (Fig. 8a). No or very little reactivity was observed for CD4+ T cells (Fig. 8a).

[0074] In final analyses, we examined whether the observed JBT19-reactivity of expanded lymphocytes was associated with any cytotoxic impact on JBT19 cells. We prepared JBT19 cells stably expressing the fluorescent protein TurboGFP (Fig. 8b, upper panel). We then co-cultured these cells with K-562- or JBT19-activated (primed) and expanded lymphocytes and determined the content of TurboGFP -JBT 19 cells in the cell co-culture as previously described (Stakheev D, Taborska P, Strizova Z, Podrazil M, Bartunkova J, Smrz D (2019) Scientific reports 9:4761 doi: 10.1038 / s41598-019-41182-5). We found that in the absence of lymphocytes, TurboGFP-JBT19 cells expanded (Fig. 8b, lower panel). However, when the cells were co-cultured with the expanded lymphocytes, their growth in culture was either arrested or the cells were eliminated from the cell co-culture (Fig. 8b, lower panel). These data showed that the expanded lymphocytes had a cytotoxic effect on 2D cultured JBT 19 cells. These data demonstrate that the new UPS cell line is immunogenic for both allogeneic and autologous lymphocytes. This immunogenicity thus opens up possibilities for the use of JBT19 cells in research investigating immunotherapeutic interventions, or their combination with other therapeutic procedures, in the treatment of UPS.

[0075] Detection of inflammatory cytokines, IFN gamma and / or TNF alpha, via changes in JBT19 cell phenotype

[0076] The presence of the inflammatory cytokines IFN gamma and / or TNF alpha could be detected by changes in the JBT19 cell phenotype. To elicit changes in the phenotype of JBT19 cells, IFN gamma, TNF alpha (PeproTech, Rocky Hill, NJ, USA or R&D Systems, Minneapolis, MN, USA), and IFN alpha (Abeam, Cambridge, UK) were used. Adherent JBT19 cells were cultured in the presence of IFN gamma (IFNy, 200 ng / ml), TNF alpha (TNFa, 20 ng / ml), IFN alpha (IFNa, 200 ng / ml), or their combinations (IFNy+TNFa, IFNy+INFa) for 2 days and then analyzed by flow cytometry for expression of selected markers. For cell surface or intracellular expression of the markers, adherent JBT19 cells were harvested by trypsinization and washed with cell culture medium and PBS with 2 mM EDTA (PBSE). Cells were stained as described in Taborska P, Stakheev D, Svobodova H, Strizova Z, Bartunkova J, Smrz D (2020) Cancers 12 doi: 10.3390 / cancersl2123766, with the following fluorescently labeled antibodies: CD274(PD-L1)-APC (clone MIH3), HLA-ABC(MHC-I)- PE (clone W6 / 32), HLA-DP, DQ, DR(MHC-II)-APC (clone Tti39), CD95(Fas)-APC (clone DX2), CD47-APC (clone CC2C6) (BioLegend, San Diego, CA), fibroblast activation protein a(FAP)-PE (clone 427819) (R&D Systems, Minneapolis, MN, USA), CD44-PE (clone MEM-263) (Exbio, Prague, Czech Republic), and IL-8-APC (clone 8CH) (Thermo Scientific, Waltham, MA, USA).

[0077] Phenotypic characterization was performed using flow cytometry. Analyses revealed that 2-day exposure of JBT19 cells to IFN gamma not only enhances the surface expression of the antigen- presenting receptor MHC-I but also enhances the expression of PD-L1 (Fig. 9, top left and middle panels and bottom left and middle graphs), thus presumably promoting anti-PD-l / PD-Ll -mediated resistance of JBT19 tumors. Moreover, the impact of IFN gamma on the expression of antigen- presenting receptor MHC-I is also elicited by other cytokines, TNF alpha or IFN alpha, as also determined by flow cytometry after 2-day exposure to these cytokines (Fig. 9, top left panel and bottom left graph). On the other hand, these two cytokines do not promote the expression of PD-L1. However, TNF alpha is still capable of further promotion of IFN gamma-mediated enhancement of PD-L1 expression (Fig. 9, top middle panel and bottom middle graph). JBT19 cells are negative for the surface expression of MHC-II receptor once cultured in the cell culture medium. However, their 2-day treatment with IFN gamma induces, as determined by flow cytometry, its de novo expression on their surface, virtually turning MHC-II-negative JBT19 cells into MHC-II+ JBT19 cells (Fig. 9, top right panel and bottom right graph). In addition, although the other two cytokines, TNF alpha and IFN alpha, are not able to de novo induce the surface expression of MHC-II receptor after the 2-day exposure, they can differentially impact the IFN gamma-induced de novo surface expression of MHC-II receptor - TNF alpha promotes this expression, IFN alpha does the opposite (Fig. 9, top right panel and bottom right graph).

[0078] The impact of the 2-day-elicited IFN gamma-induced de novo surface expression of MHC-II receptor is sustainable for 2 days after removing this cytokine, as determined by flow cytometry (Fig. 10, top left panel and bottom left graph). A similar result is also true for the 2-day-elicited IFN gammainduced enhanced surface expression of the antigen-presenting receptor MHC-I, as determined by flow cytometry (Fig. 10, top right panel and bottom right graph).

[0079] The sustainability of the cytokine-induced enhanced surface expression of PD-L1 in JBT19 cells is different. Unlike MHC-I or MHC-II receptors, the removal of the cytokine after 2-day-elicited IFN gamma-induced enhanced surface expression of PD-L1 leads to a partial reversion of the enhancement in 2 days as determined by flow cytometry (Fig. 11), showing the plasticity of the cells in the PD-L1 surface expression in response to external inflammatory stimuli, such as IFN gamma.

[0080] Apart from the above-noted surface molecules, IFN gamma and TNF alpha also enhance the surface expression of CD47 (2-day treatment) (Fig. 12, top left panel and top left graph) and CD44 (8-day treatment) (Fig. 12, bottom right graph), and IFN gamma enhances the expression of Fas (CD95) (2- day treatment) (Fig. 12, top middle panel and top right graph). No observable changes these two cytokines elicit in the surface expression of FAP (2-day treatment) (Fig. 12, top right panel and bottom left graph), which was used as a negative control in the experiments. In addition to the surface expression of the phenotypic markers, JBT19 cells de novo also produce the cytokine IL-8 in response to 7-day exposure to TNF alpha, as determined by intracellular staining for this cytokine and flow cytometry analysis (Fig. 13).

[0081] These data demonstrate that the new UPS cell line is sensitive to the addition of extracellular inflammatory cytokines, which leads to changes in its phenotype. This property of the JBT19 cells thus opens possibilities for using JBT19 cells in detecting inflammatory cytokines in biological samples. Functionality of the changed JBT19 cell phenotype after exposure of JBT19 cells to IFN gamma

[0082] To investigate the functionality of the changed phenotype of JBT19 cells that was induced by the inflammatory cytokines, allogeneic JBT19-reactive lymphocytes were prepared as described in the section “Preparation of autologous and allogeneic JBT19-reactive T cells and NK cells.” For the preparation of the JBT19-reactive lymphocytes, 2-day IFN gamma-treated JBT19 cells (IFNy, 200 ng / ml) were used to also stimulate (prime) the expansion of CD4+ T cells in the cell culture via the de Movo-induced surface expression of MHC-II receptor in the treated JBT19 cells. The prepared lymphocytes were then stimulated with JBT19 cells not treated or 2-day treated with IFN gamma (200 ng / ml), and the percentage of reactive (specific) lymphocytes, CD4+ or CD8+ T cells, were detected as the percentage of cells producing the pro-inflammatory cytokines TNF-alpha and IFN-gamma, which were detected by intracellular labeling using fluorescently labeled antibodies. As shown, JBT19 cells not treated with IFN gamma (JBT19 stim) induce a notable production of only IFN gamma in CD4+ T cells as compared with unstimulated (No stim) control (Fig. 14, top middle and left panel and middle graph below). No TNF alpha nor TNF alpha / IFN gamma-producing CD4+ T cells is detected after stimulation with JBT19 cells not treated with IFN gamma (JBT19 stim) as compared with unstimulated (No stim) control (Fig. 14, top middle and left panels and left and right graphs below). However, stimulation with JBT19 cells treated for 2-days with IFN gamma (IFNy-treated JBT19-stim) induces a notable production of IFN gamma, TNF alpha, or IFN gamma / TNF alpha in CD4+ T cells as compared with both unstimulated (No stim) control or cells stimulated with untreated JBT19 cells (JBT19 stim) (Fig. 14, top panels and graphs below). The IFN gamma treatment of JBT19 cells is even more pronounced in the reactivity of CD8+ T cells in the cell culture of the prepared lymphocytes. As shown, stimulation of the lymphocytes with 2-day IFN gamma-treated JBT19 cells (IFNy-treated JBT19-stim) induce remarkably much higher production of IFN gamma, TNF alpha, or IFN gamma / TNF alpha in CD8+ T cells (Fig. 14, bottom right panel and graphs below) as compared with stimulation of the lymphocytes with untreated JBT19 cells (JBT19 stim) or unstimulated lymphocytes (No stim) (Fig. 14, bottom middle and left panels and graphs below). These data demonstrate that the new UPS cell line is not only sensitive to the addition of extracellular inflammatory cytokines, which leads to changes of its phenotype, but that the changed phenotype is functional. This property of the JBT19 cells thus opens possibilities for the use of JBT19 cells in detecting inflammatory cytokines in biological samples also through the analyses of the functionality of the cytokine-elicited phenotypes.

Claims

CLAIMS1. Cells derived from undifferentiated pleomorphic sarcoma that express PD-L1 and collagen and are deposited in the European Collection of Authenticated Cell Cultures under accession number 22113001.

2. Use of the cells according to claim 1 for testing immunotherapeutics in vitro or ex vivo.

3. Use of the cells according to claim 1 for testing immunotherapeutics for adoptive cell immunotherapy and immunotherapy with cells of the immune system in vitro or ex vivo.

4. Use of the cells according to claim 1 in immunotherapeutic research in vitro or ex vivo as target cells.

5. Use of the cells according to claim 1 for ex vivo production of immune cells reactive to the cell line of claim 1 from allogeneic and autologous lymphocytes.

6. Use according to claim 5, wherein the immune cells reactive to the cell line of claim 1 are NK cells, CD8+ T cells and CD8+NKT-like cells.

7. Use of the cells according to claim 1 for xenotransplantation and / or xenoimplantation / xenografting in animals, in particular mice, for non-therapeutic and non-diagnostic purposes.

8. Cells according to claim 1 for use for xenotransplantation and / or xenoimplantation / xenografting in animals, in particular mice, for therapeutic and / or diagnostic purposes.

9. A method for preparing a tumor cell line from a tumor tissue, comprising the steps of: disintegrating the tumor tissue to form a cell suspension, adding an anti-TNF-a antibody to the cell suspension after disintegration, and further culturing the cell suspension under adherent conditions.

10. The method according to claim 9, wherein at passage or at exchange of medium, medium added to the cell suspension contains anti-TNF-a antibody.

11. An in vitro method of detecting inflammatory cytokines in a biological sample, comprising:a step of contacting the cells according to claim 1 with the biological sample, and a subsequent step of detecting expression of MHC-II and / or IL-8, and / or detecting the extent of MHC- I, PD-L1, CD47, Fas (CD95) and / or CD44 expression in the cells according to claim 1.

12. An in vitro method of detecting inflammatory cytokines in a biological sample, comprising: a step of contacting the cells according to claim 1 with the biological sample, and a subsequent step of detecting functionality of MHC-II and / or IL-8, and / or detecting the extent of functionality MHC-I, PD-L1, CD47, Fas (CD95) and / or CD44 in the cells according to claim 1, in order to determine the expression of MHC-II and / or IL-8, and / or to determine the extent of expression of MHC-I, PD-L1, CD47, Fas (CD95) and / or CD44.

13. The method according to claim 11 or 12, wherein the biological sample is selected from serum, exudate, disintegrated tumor tissue.

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