Multiexpresividad: escenario para la convergencia y los enfoques relacionales

4.3.1. Generation of PD-L1+ _{AML target cell lines}

Screenings of CD33 expression levels in AML patient samples report a significant but heterogeneous upregulation of the myeloid differentiation antigen with high variation between

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patients and leukemic subpopulations.79 Thus, two AML cell lines with different CD33 expression levels were selected as target cell lines. MOLM-13 cells were identified to express high (CD33bright) and OCI-AML3 low (CD33dim_{) CD33 levels, however, both of them lack PD-L1 on the cell surface} (Figure 9). The second known ligand of PD-1, i.e. PD-L2, was not detected on MOLM-13 but on OCI-AML3 cells.

Figure 9: Antigen expression by selected AML cell lines.

Flow cytometric evaluation of CD33, PD-L1 and PD-L2 expression on parental MOLM-13 and OCI-AML3 cells. The black line indicates unspecific staining by the isotype control.

It is reported in vitro and in vivo that IFN-γ induces the upregulation of PD-L1 on AML cells.189- 191

Thus, MOLM-13 and OCI-AML3 cells were incubated with 100 ng/µl IFN-γ and after 24 h PD-L1 expression was analyzed by flow cytometry. However, with this strategy, no significant PD-L1 induction could be triggered (data not shown). Thus, stable PD-L1 expression on MOLM-13 and OCI-AML3 cells was ensured by retroviral transduction of the parental cell lines f. This strategy guaranteed the maintenance of a stable cell background for functional assays and enabled the selection of cell lines with distinct PD-L1 expression levels. MOLM-13 cells were transduced with a pMXs vector containing the full-length human PD-L1 cDNA sequence, which yielded a transduction efficiency of 11% (Figure 10). Subsequently, PD-L1+ cells were separated by FACS sorting, and by dilution cloning homogeneous cell lines were raised from single cells. In total, the PD-L1 expression level of 78 clones was assessed by flow cytometry, and the five shown

f _{Retroviral transduction and FACS sorting were performed by Felicitas Rataj and Constanze Heise, Laboratory} of Sebastian Kobold, Clinical Pharmacology, Department of Internal Medicine IV, Klinikum der LMU München, Germany

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in Figure 10 B were cryoconserved for further analysis. Clone 56 was selected for the present study since it exhibited similar expression levels for PD-L1 and CD33. It is hereafter referred to as MOLM-13:PD-L1 cell line. Characterization of MOLM-13 and MOLM-13:PD-L1 cells confirmed similar CD33 levels and homogeneous PD-L1 expression on MOLM-13:PD-L1 cells (Figure 10 C). According to the same procedure, OCI-AML3:PD-L1 cells were generated.

Figure 10: Generation of PD-L1+ _{MOLM-13 cells by transduction and single-cell cloning.}

(A) Flow cytometric analysis of PD-L1-transduced bulk MOLM-13 cells in comparison to parental MOLM-13 cells. (B) PD-L1+ _{single cell clones with different PD-L1 expression levels. (C) CD33 and PD-L1 expression of} selected MOLM13:PD-L1 clone 56 in comparison to parental MOLM-13 cells. The black line indicates unspecific staining.

All cell lines were quantified regarding their surface antigen density (Table 17). Notably, CD33 and PD-L1 expression levels were in a similar range on MOLM-13:PD-L1 cells, whereas OCI- AML3:PD-L1 cells displayed a significantly higher density of PD-L1 compared to CD33.

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Table 17: Surface antigen density of AML target cell lines as determined by QIFIKIT.

Table summarizes mean values of surface antigen density calculated from 3-4 experiments. Errors indicate SEM.

CD33 PD-L1

MOLM-13 _69.2x103 _{± 8.2x10}3 -

MOLM-13:PD-L1 _78.3x103 _{± 4.5x10}3 _99.3x103 _{± 9.7x10}3

OCI-AML3 _3.0x103 _{± 0.6x10}3 -

OCI-AML3:PD-L1 _3.3x103 _{± 1.3x10}3 _121.9x103 _{± 7.9x10}3

4.3.2. Generation of stable PD-L1+_{, CD33}+ _{and CD33}+_PD-L1+ _{target cell lines}

Since PD-L1 can be ubiquitously upregulated on cells in the presence of proinflammatory cytokines, for the evaluation of potential on-target off-leukemia effects of CiTE and sctb a target cell system was required that represents this cell population of the body.107 _{To this end, the Flp-} In™ 293T-Rex system was used to raise PD-L1+, CD33+ as well as CD33+PD-L1+ cell lines of the same cell background. Stable cell lines were grown under hygromycin selection pressure within several weeks. The resulting HEK293:PD-L1, HEK293:CD33 and HEK293:CD33:PD-L1 cell lines were confirmed to homogeneously express the integrated antigens and PD-L1 and CD33 levels were quantified (Table 18). Notably, PD-L1 expression on HEK293:PD-L1 cells was determined to be similar to HEK293:CD33:PD-L1 cells. Basal antigen expression of all three cell lines could be increased by the addition of tetracycline. Yet, induced cell lines were exclusively applied for binding studies, whereas cell lines with antigen expression in a physiologically more relevant range were utilized for functional assays.

Table 18: Surface antigen density of Flp-In™T-Rex-derived cell lines as determined by QIFIKIT.

Table summarizes mean values of surface antigen density calculated from 3-4 experiments. Errors indicate SEM.

CD33 PD-L1 HEK293:PD-L1 - _8.8x103 _{± 1.8x10}3 HEK293:PD-L1_ind. - _348.2x103 _{± 13.8x10}3 HEK293:CD33 _{22.5 x10}3 _{± 7.8x10}3 - HEK293:CD33_ind. _235.7x103 _{± 3.4x10}3 - HEK293:CD33:PD-L1 _15.4x103 _{± 0.6x10}3 _8.5x103 _{± 0.9x10}3

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