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. 2010 Aug 19;116(7):1124-31.
doi: 10.1182/blood-2009-12-255125. Epub 2010 May 14.

Interferon-gamma and tumor necrosis factor-alpha induce an immunoinhibitory molecule, B7-H1, via nuclear factor-kappaB activation in blasts in myelodysplastic syndromes

Asaka Kondo  1 , Taishi Yamashita, Hideto Tamura, Wanhong Zhao, Takashi Tsuji, Masumi Shimizu, Eiji Shinya, Hidemi Takahashi, Koji Tamada, Lieping Chen, Kazuo Dan, Kiyoyuki Ogata
Affiliations

Interferon-gamma and tumor necrosis factor-alpha induce an immunoinhibitory molecule, B7-H1, via nuclear factor-kappaB activation in blasts in myelodysplastic syndromes

Asaka Kondo et al. Blood. .

Abstract

During disease progression in myelodysplastic syndromes (MDS), clonal blasts gain a more aggressive nature, whereas nonclonal immune cells become less efficient via an unknown mechanism. Using MDS cell lines and patient samples, we showed that the expression of an immunoinhibitory molecule, B7-H1 (CD274), was induced by interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha) on MDS blasts. This induction was associated with the activation of nuclear factor-kappaB (NF-kappaB) and nearly completely blocked by an NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC). B7-H1(+) MDS blasts had greater intrinsic proliferative capacity than B7-H1(-) MDS blasts when examined in various assays. Furthermore, B7-H1(+) blasts suppressed T-cell proliferation and induced T-cell apoptosis in allogeneic cocultures. When fresh bone marrow samples from patients were examined, blasts from high-risk MDS patients expressed B7-H1 molecules more often compared with those from low-risk MDS patients. Moreover, MDS T cells often overexpressed programmed cell death 1 (PD-1) molecules that transmit an inhibitory signal from B7-H1 molecules. Taken together, these findings provide new insight into MDS pathophysiology. IFNgamma and TNFalpha activate NF-kappaB that in turn induces B7-H1 expression on MDS blasts. B7-H1(+) MDS blasts have an intrinsic proliferative advantage and induce T-cell suppression, which may be associated with disease progression in MDS.

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Figures

Figure 1
Figure 1
Constitutive or inducible B7-H1 expression in MDS cell lines.(A) B7-H1 mRNA expression analyzed by reverse-transcription polymerase chain reaction. Equal amounts of cDNA from 3 myelodysplastic syndromes (MDS) cell lines were amplified. Vertical lines have been inserted to indicate a repositioned gel lane. (B) B7-H1 expression analyzed by flow cytometry (FCM). The cell lines were cultured with medium alone or with IFNγ. Thick line histogram represents staining with anti–B7-H1 monoclonal antibodies; dotted line histogram represents staining with isotype-matched control Ig. (C) Effects of cytokines on B7-H1 expression in F-36P and SKM-1 cells. Data (relative fluorescence intensity [MFI]) are mean and SD of 3 experiments. Results without SD error bar indicate small SD.
Figure 2
Figure 2
Inhibition of NF-κB activation down-regulated B7-H1 expression.(A) SKM-1 cells cultured for 24 hours with medium alone, IFNγ, or IFNγ and pyrrolidine dithiocarbamate (PDTC; 100μM) were stained for NF-κB p65 (red) and nuclei (blue). The merged image showed that IFNγ induced p65 localization into the nucleus, which was inhibited by PDTC. Images were acquired on an Olympus IX71 inverted microscope using a LCPlanFI 40×/0.60 objective lens. Alexa 594 and 4'-6-diamidino-2-phenylindole were detected using Fluorescence Miler Units of U-MWIG2 and U-MNUA2 (Olympus), respectively. Images were captured using a 3CCD digital color camera (Hamamatsu Photonics) through Aquacosmos Software. (B) After the cultures in A, SKM-1 cells were separated into cytoplasmic and nuclear fractions, and their p65 contents were analyzed by Western blotting to show representative (top panel) and quantified (bottom panel) data (mean + SD) of 3 experiments. In each experiment, the band intensity of cell fractions from SKM-1 cells cultured with medium alone was defined as 1. *P = .017 and .06 (left panel) and *P = .045 and .012 (right panel) compared with “the medium” and IFNγ and PDTC, respectively. (C) SKM-1 cells cultured with or without cytokine(s) and various concentrations of PDTC were analyzed for B7-H1 expression by FCM. (D-E) Using F-36P cells expressing B7-H1 constitutively, the same experiments as in panels B and C were performed, except that no cytokines were used. Data are from 3 experiments. *P = .015 for D (left panel) and *P < .0001 (right panel) compared with the medium.
Figure 3
Figure 3
Cell proliferative potential as a function of B7-H1 expression in MDS cell lines. (A-B) Cell-cycle analysis of B7-H1+ and B7-H1− cell fractions in F-36P (A) and SKM-1 (B) cells. SKM-1 cells were cultured with IFNγ for 2 days before analyses. (C) BrdU incorporation in B7-H1+ and B7-H1− cell fractions in F-36P and SKM-1 cells. (D) The number of colonies formed by purified B7-H1+ and B7-H1− F-36P cells in the methylcellulose culture. Data in panels A through C are mean (and SD) of 3 experiments. *P < .036 for B7-H1+ versus B7-H1− cell fractions.
Figure 4
Figure 4
Effects of B7-H1 molecules expressed by blasts on T cells. (A-C) Purified, normal CD3+, CD4+, and CD8+ T cells were cultured alone or with irradiated F-36P cells in the presence of either control immunoglobulin G (IgG), anti–B7-H1 monoclonal antibody, or anti–PD-1 monoclonal antibody. Data are mean (and SD) of 3 experiments. P < .05 when data in each of the 2 columns on the right were compared with control IgG data. (A) The percentage of annexin V+ cells in each T-cell fraction. (B) The percentage of caspase-3+ cells in T cells. Data labeled control IgG were defined as 100% in each experiment. (C) T-cell proliferation determined in the 3H-thymidine incorporation assay. (D) Normal CD3+ T cells were cultured with irradiated F-36P cells that had been purified into either B7-H1+ or B7-H1− cells.
Figure 5
Figure 5
B7-H1 expression on patient blasts. (A) The percentages of B7-H1+ cells in blasts from freshly isolated bone marrow samples. (B) The induction of B7-H1 expression on blasts from 7 patients. The cells had been cultured for 2 days with IFNγ and/or TNFα. (C) Purified CD34+ blasts from a patient (no. 38) were incubated with no additive (Medium), PDTC (100μM), IFNγ, or IFNγ and PDTC. After incubation, the blasts were separated into cytoplasmic and nuclear fractions. The NF-κB p65 contents in each fraction were analyzed using Western blotting to show representative (top panel) and quantified (bottom panel) data (mean + SD) of 2 experiments. In each experiment, the band intensity of cell fractions from blasts that had been cultured with no additive was defined as 1. (E) The inhibitor of NF-κB, PDTC, blocked B7-H1 induction by IFNγ on patient blasts. Purified CD34+ blasts from patients were cultured as indicated, and their B7-H1 expression was analyzed.
Figure 6
Figure 6
Functional aspects of B7-H1-expressing blasts from patients. (A) Cell cycle of purified CD34+ blasts as a function of B7-H1 expression. Freshly isolated blasts that markedly expressed B7-H1 molecules (from patient no. 36) and blasts cultured with IFNγ and TNFα to induce B7-H1 expression (from patient no. 10 and patient no. 40) were analyzed. B7-H1+ (■) and B7-H1− (□) cells are indicated. (B) B7-H1–expressing patient blasts were cultured with normal T cells in the presence of control IgG or anti–B7-H1 monoclonal antibodies or medium alone. Data are mean (and SD) of 3 experiments; in each experiment using a different patient sample, data labeled control IgG were defined as 100%. *P = .026 compared with control IgG.
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