The Effect of Acute Myeloid Leukemia Cells Expressing PD-L2 Co-Stimulatory Molecule on Helper T Cell Activation

Abstract

The binding of PD-1 to its ligands (PD-L1 and PD-L2) inhibits and/or regulates T cell responses. Myeloid leukemia cells can express both co-activatory and co-inhibitory molecules simultaneously. The function of PD-L1 in regulation of antitumor responses is well-known. Although under physiological conditions, PD-L2 is expressed on mature antigen presenting cells (APCs), it can also be highly expressed on monocytic leukemia that is composed of immature blasts. In this study, THP-1 was used as monocytic leukemia cell line and it resembles to primary monocytes. Monocytes were cultured in vitro to differentiate, while THP-1 cells were differentiated into monocyte/macrophage-like cells with protein kinase C activator, phorbol 12-myristate 13-acetate (PMA) and these cells were called “pTHP-1 cells”. Then, the expression of PD-1 ligands and B7 molecules were modulated and their effect on T helper responses was evaluated. Primary monocytes used as controls were maturated in suspension in vitro. By this way, the expressions of B7 molecules (especially PD-L2) were modulated and morphologically, macrophage-like cell type was obtained. However, because PD-L1 could not have been upregulated on pTHP-1 cells and cultured monocytes, THP-1 and pTHP-1 were stimulated with IFN-γ as a critical cytokine in anti-tumor responses. These cells were co-cultured with Th cells or peripheral blood mononuclear cells (PBMCs). In the co-cultures, the first signal was provided by anti-CD3 mAb and the second signal was provided by co-stimulatory molecules expressed on monocyte/macrophage-like cells. Although THP-1 cells and monocytes resemble to each other, they behaved differentially. It was also found that the effect of THP-1 cells on Th cells was generally maintained through co-stimulatory mechanisms. Upon IFN-γ treatment, monocytes gained less stimulatory character. When PD-L2 molecule was blocked on IFN-γ-treated and control monocytic cells, it led to increase in the proliferation of Th cells and promote Th1 differentiation. Upon PD-L2 blockade, especially in the co-cultures with control monocytes, the production of Th2-related cytokines were also increased. To confirm the results, HEK293T cells were transfected with PD-L1, PD-L2 and CD86 genes. No Treg differentiation or T cell exhaustion was observed. On the other hand, blockade of PD-L2 molecule promoted immune activation. Consequently, in this study, the immune modulatory effects of PD-L2 on primary monocytes cells and on monocytic leukemia cells were compared and novel findings were obtained.