Interferon-γ and Tumor Necrosis Factor-α Induce An Immunoinhibitory Molecule, B7-H1, Via NfκB Activation in Blasts of Myelodysplastic Syndromes.

B7-H1 (CD274) is a costimulatory/coinhibitory molecule that plays a crucial role in T cell regulation in various immune responses. B7-H1 expression is detected not only on professional antigen-presenting cells but also on some tumor cells. It has been reported that B7-H1 molecules on tumor cells inhibit the proliferation of tumor-specific cytotoxic T-lymphocytes and are associated with poor prognosis in patients with some tumors such as renal and breast cancers. In this study, we investigated whether functional B7-H1 molecules are expressed on blasts from myelodysplastic syndromes (MDS) and, if so, are associated with pathophysiology in specific immune evasion in MDS.

(1) First, we analyzed the expression of B7-H1 mRNA and protein in MDS cell lines (OIH-1, SKM-1, and F36P) using reverse transcription-PCR and flow cytometry (FCM), respectively. Although F36P cells alone expressed B7-H1 molecules at mRNA and protein levels, interferon (IFN)-γ and more efficiently a combination of IFN-γ and tumor necrosis factor (TNF)-α enhanced or induced B7-H1 expression in all of these cell lines. Notably, the combination of IFN-γ and TNF-α induced nuclear factor (NF) γB activation of MDS cell lines, and the blocking of NFκB activation by pyrrolidine dithiocarbamate (PDTC) or NFκB inhibitor peptide turned off the B7-H1 induction. (2) To investigate whether B7-H1+ MDS blasts have a proliferative advantage, we compared the cell cycle and colony formation between B7-H1+ and B7-H1– cell fractions in F36P cells. B7-H1+ cells had fewer G0/G1-phase cells and more G2/M-phase cells compared with B7-H1– cells. Furthermore, purified B7-H1+ cells formed more colonies than B7-H1– cells in the colony-forming assay using the methylcellulose. (3) We investigated the immunomodulatory effects of B7-H1+ blasts on T cells. When normal CD3+ T cells were cultured with or without irradiated B7-H1+ F36P cells, the B7-H1+ F36P cells had markedly increased percentages of T cells showing apoptosis. The blocking antibody for B7-H1 or PD-1 inhibited the T-cell apoptosis induced by B7-H1+ cells, decreased the percentage of caspase-3+ cells in T cells, and increased T-cell proliferation. (4) We then examined B7-H1 expression on blasts from 41 MDS patients, 20 patients with acute myeloid leukemia transformed from MDS (AML-MDS), and 10 hematologically normal individuals using FCM. B7-H1+ cases, in whom more than 5% of blasts expressed B7-H1 molecules, were observed in 6 of 14 (43%) patients with refractory anemia with excess blasts (RAEB), 6 of 12 (50%) with RAEB in transformation (RAEB-t), and 4 of 20 (20%) AML-MDS patients, but in none of 15 RA patients and normal individuals. Similar to the data obtained using MDS cell lines, B7-H1 expression on blasts purified from MDS patients was induced by stimulation with IFN-γ or TNF-α, and more strikingly by their combination. PDTC inhibited the induction of B7-H1 expression. The percentages of G0/G1-phase cells were lower and those of G2/M-phase cells higher in B7-H1+ blasts than in B7-H1– blasts from patients. Furthermore, in allogeneic coculture, in which B7-H1+ blasts from an MDS patient and CD3+ T cells from healthy volunteers were cultured, T-cell proliferation was significantly augmented by blocking B7-H1 molecules. (5) Finally, we investigated the expression of PD-1, a counterreceptor of B7-H1, on circulating T cells in 34 MDS patients. PD-1 expression on CD3+, CD4+, and CD8+ T cells in MDS patients was significantly higher than that in healthy volunteers.

Taking the results together, in MDS: 1) B7-H1 expression on blasts occurs more often in the advanced disease stages; 2) B7-H1 expression on blasts may be induced by cytokines derived from the bone marrow environment via NFκB activation; 3) cell cycle and proliferation were more active in B7-H1+ blasts than in B7-H1– blasts; and 4) B7-H1 molecules on blasts suppress T-cell immunity. These findings indicate that B7-H1 molecules on blasts may be involved in the pathophysiology of MDS.

No relevant conflicts of interest to declare.