ISSN 1866-8836
Клеточная терапия и трансплантация

BS-08. Development of a PD-L1 and Tim-3 expression model using human myeloid cell lines for investigating the activity of small molecule inhibitors of immune checkpoints signaling pathways

Dina A. Senichkina1, Alena I. Shakirova1, Olga S. Epifanovskaya1, Ivan N. Gaponenko1, Yekaterina V. Belotserkovskaya2, Anna B. Malyshecheva2, Kirill V. Lepik1, Ivan S. Moiseev1

1 RM Gorbacheva Research Institute, Pavlov University, St. Petersburg, Russia
2 Institute of Cytology, St. Petersburg, Russia

Contact: Dina A. Senichkina, e-mail:

doi 10.18620/ctt-1866-8836-2023-12-3-1-176


High-risk myelodysplastic syndrome (MDS) is a prognostically unfavorable group of clonal diseases of the hematopoietic tissue with a low overall survival of not more than two years with existing treatments and a high risk of transformation into acute myeloid leukemia (AML). Among possible targets for targeted MDS therapy, immune checkpoint receptors and ligands (IC) have potential. ICT expression of PD-1/PD-L1 and Tim-3/Gal-9 has been shown in myeloid neoplasms, including MDS, on tumor and immune microenvironment cells (Yang, X., 2002). It is important to note that the literature describes the occurrence of Tim-3 expression after unsuccessful treatment with anti-PD1 antibodies with the loss of the last marker on the surface of immune/blast cells (Koyama, S. et al, 2016). The aim of this study was to create a model with increased expression of PD-L1 and human cell line-based Tim-3 of leukemic genesis for the study of small molecule inhibitors of ICT signaling pathways with myeloid neoplasia.

Materials and methods

Initial testing of basal level of an expression of PD-L1 and Tim-3 was carried out on cellular lines: THP-01, Caco2, A549, HL-60, OCI-AML-2 cultivated in standard conditions. The lines used in the work were provided by the Russian Collection of Cell Cultures of the Institute of Scientific Centers of the Russian Academy of Sciences. The following compounds were used to induce the expression of ICT receptors: for PD-L1, interferon gamma (Ingaron, Russia), for Tim-3, bacterial lipopolysaccharides (LPS) (Pyrogenal, Russia). Specific marker expression was evaluated by flow cytofluorimetry by staining cells with a cocktail of anti-TIM-3 monoclonal antibodies (CD366-APC), and anti-PD-L1 (CD274-PE-Cy7), counterstained by a vital dye DRAQ7.


Pre-screening showed no expression of CD274 and CD366 on all tested lines. It was shown that we need to induce the expression of Tim-3 and PD-L1 in these model cells. Under the conditions of culturing THP cells with different interferon concentrations (2.5 to 750 ng/mL) for 24 hours, the concentration of 50 ng/mL was found to be optimal. At this concentration, an increased surface expression of PD-L1 was observed in THP-1 cells (up to 80%) and in OCI-OCI-AML-2 cells (up to 30%) with high survival parameters. The effect of PD-L1 expression could not be achieved in HL-60 cells. Incubation for 2 hours of THP-1 cells in the presence of LPS (25 ng/ml-10 μg/mL) caused an increase of Tim3 expression to 6-8% without reducing viability (90%). At the same time, during long-term cultivation (4-24 hours), including in the presence of gamma interferon (50 ng/ml), Tim-3 levels did not differ from the control.


During the study, a method for inducing PD-L1 expression by tumor cell lines was successfully developed, suitable for testing the effects of inhibition of signaling pathways of this receptor. The concentration of interferon-gamma of 50 ng/ml was optimal for inducing PD-L1 expression in 80% of cells. A further increase in PD-L1 inducer concentration is not appropriate because it may interfere with the identification of compounds having only moderate effects on PD-L1 expression (Spangenberg H. et. al, 2021). The Tim-3 stable expression induction method needs further optimization in THP-1 cells.


Yang X., Ma L, Zhang X et al. Targeting PD-1/PD-L1 pathway in myelodysplastic syndromes and acute myeloid leukemia. Exp Hematol Oncol 11, 11 (2022).

Koyama S, Akbay E, Li Y et al. Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints. Nat Commun 7, 10501 (2016).

Spangenberg SH, Zavareh RB, Lairson LL. Protocol for high-throughput compound screening using flow cytometry in THP-1 cells, STAR Protocols (2021).


PD-L1, Tim-3, THP-1, immune checkpoints, acute myeloid leukemia, myelodysplastic syndrome, inhibitors, AML, MDS.

Supplement 12-3

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doi 10.18620/ctt-1866-8836-2023-12-3-1-176

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