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. 2018 Dec 5;8(2):e1546544.
doi: 10.1080/2162402X.2018.1546544. eCollection 2019.

Pomalidomide increases immune surface marker expression and immune recognition of oncovirus-infected cells

David A Davis  1 , Prabha Shrestha  1 , Ashley I Aisabor  1 , Alexandra Stream  1 , Veronica Galli  2 , Cynthia A Pise-Masison  2 , Takanobu Tagawa  1 , Joseph M Ziegelbauer  1 , Genoveffa Franchini  2 , Robert Yarchoan  1
Affiliations

Pomalidomide increases immune surface marker expression and immune recognition of oncovirus-infected cells

David A Davis et al. Oncoimmunology. .

Abstract

Most chronic viruses evade T-cell and natural killer (NK) immunity through downregulation of immune surface markers. Previously we showed that Pomalidomide (Pom) increases surface expression of major histocompatibility complex class I (MHC-I) in Kaposi sarcoma-associated herpesvirus-infected latent and lytic cells and restores ICAM-1 and B7-2 in latent cells. We explored the ability of Pom to increase immune surface marker expression in cells infected by other chronic viruses, including human T-cell leukemia virus type-1 (HTLV-1), Epstein-Barr virus (EBV), human papilloma virus (HPV), Merkel cell polyoma virus (MCV), and human immunodeficiency virus type-1 (HIV-1). Pom increased MHC-1, ICAM-1, and B7-2/CD86 in immortalized T-cell lines productively infected with HTLV-1 and also significantly increased their susceptibility to NK cell-mediated cytotoxicity. Pom enhancement of MHC-I and ICAM-1 in primary cells infected with HTLV-1 was abrogated by knockout of HTLV-1 orf-1. Pom increased expression of ICAM-1, B7-2 and MHC class I polypeptide related sequence A (MICA) surface expression in the EBV-infected Daudi cells and increased their T-cell activation and susceptibility to NK cells. Moreover, Pom increased expression of certain of these surface markers on Akata, Raji, and EBV lymphoblastic cell lines. The increased expression of immune surface markers in these virus-infected lines was generally associated with a decrease in IRF4 expression. By contrast, Pom treatment of HPV, MCV and HIV-1 infected cells did not increase these immune surface markers. Pom and related drugs may be clinically beneficial for the treatment of HTLV-1 and EBV-induced tumors by rendering infected cells more susceptible to both innate and adaptive host immune responses.

Keywords: CD86; ICAM-1; Natural killer (NK) cells; T cells; epstein barr virus (EBV); human T lymphotropic virus type 1 (HTLV-1); ikaros (IKZF1).; interferon regulatory factor 4 (IRF4); major histocompatibility complex class I (MHC-I); pomalidomide.

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Figures

Figure 1.
Figure 1.
Pom increases MHC-I, ICAM-1 and B7-2 surface expression in HTLV-1 infected MT-2 cells but not in HTLV-1 infected TLOM1 cells. Indicated MT-2 cells (A,B) or TLOM1 cells (C,D) were treated for 3 days and then analyzed for surface expression markers. (A,C) Shown are representative histograms of DMSO control (solid black line) and 10 µM Pom treated (dashed line) MT-2 (A) or TLOM1 (C) cells for MHC-I, ICAM-1, and B7-2. Isotype controls are shown shaded in grey. (B,D). Fold change in MHC-I, ICAM-1, and B7-2 for MT-2 cells (B) or TLOM1 cells (D) treated for three days with 0, 1 and 10 µM Pom. Shown are the averages ± the standard deviations of 4 separate experiments for MT-2 cells and 3 separate experiments for TLOM1 cells. Asterisks indicate p values as follows compared to DMSO control: * p < 0.05, **p < 0.01.
Figure 2.
Figure 2.
Effect of Pom on cellular IKZF1, IRF4 and MHC-I in MT-2 cells and TLOM1 cells. Immunoblots for IKZF1, IRF4 and beta actin in MT-2 cells (A) or TLOM1 cells (B) treated for 10 days with 10 µM Pom or DMSO control. Relative protein levels for IRF4 and MHC-I were determined based on the loading controls (beta-actin or tubulin) using the Licor system and the relative values are shown below the images.
Figure 3.
Figure 3.
Pom increases NK cell-mediated cytotoxicity against MT-2 HTLV-1-producing cells but not of TLOM1 HTLV-1 nonproducing cells. MT-2 cells (A) or TLOM1 cells (B) were treated with DMSO control or 1 µM Pom for 5 days and then assayed for NK cell-mediated cytotoxicity using YTS effector cells with effector-to-target ratios ranging from 0.25:1 to 5:1. The data represent the average of 3 independent experiments ± the standard deviation. Asterisks indicate p values as follows compared with the DMSO control: * p < 0.05, ** p < 0.01. In these experiments, expression of B7-2 increased from 2.9–3.6 fold in MT-2 cells treated for 5 days with 1 µM Pom but was not increased in TLOM-1 cells.
Figure 4.
Figure 4.
Pom increases MHC-I and ICAM-1 expression in WT HTLV-1 but not orf-I/P12 knockout HTLV-1-infected CD4+ primary T-cells. CD4+ primary T-cells infected with WT HTLV-1 (A) or orf-I knockout HTLV-1 (B) were treated with DMSO control (solid black line) or 1 µM pomalidomide (dashed line) for four days. Cells were then analyzed by flow cytometry for MHC-I (left panel) and ICAM-1 (right panel) expression. The isotype control is shown shaded in grey. Shown are representative results from two separate experiments for WT infected cells but only one experiment for orf-I knockout-cells due to limitation of available cells.
Figure 5.
Figure 5.
Pom increases MHC-I surface expression in EBV-infected akata cells but does not increase ICAM-1 or B7-2 expression. Akata cells were treated for 2 days with DMSO control, 1 µM Pom or 10 µM Pom. (A) Representative histograms of each surface marker for DMSO control (solid line), 1 µM Pom (dashed line) or 10 µM Pom (dotted line) treated cells. The isotype controls are shown shaded in grey. (B) The fold change in MHC-I, ICAM-1, and B7-2 in Akata cells treated for three days with 0, 1 and 10 µM Pom. Shown are the averages ± standard deviations of 4 separate experiments for MHC-I, (only 3 experiments at 10 µM) and 3 separate experiments for ICAM-1 and B7-2. Asterisks indicate p values as follows compared to DMSO control: * p < 0.05, ** p < 0.01. (C,D) Nuclear extracts were prepared 2 days after treatment and analyzed for IKZF1 (C) and IRF4 (D) by immunoblot. β-actin was measured as a loading control. (E) Immunoblot for MHC-I with tubulin as a loading control from cytoplasmic extracts from Akata cells treated for 2 days with 0, 0.1, 1.0 and 10 µM Pom. In C, D and E, the relative levels of IKZF1, IRF4, and MHC-I are indicated under the blots and are relative to the DMSO treated controls using the Licor system.
Figure 6.
Figure 6.
Pom increases ICAM-1, B7-2, and MICA expression in EBV-infected Daudi cells. Daudi cells were treated for two days with DMSO control or Pom (0, 0.1 1, or 10 µM). Shown are representative histograms of each surface marker for (A) ICAM-1, (B) B7-2, and (C) MICA for cells treated with DMSO (solid line) or 1 µM Pom (dashed line). The isotype control is shown in grey. The average fold changes for these markers are shown in the bar graphs to the right. Shown on the graphs are the averages ± standard deviation from five independent experiments for ICAM-1 and B7-2, and three independent experiments for MICA. Asterisks indicate p values as follows compared to DMSO control: *p < 0.05, **p < 0.01, and ***p < 0.005. (D) Levels of IKZF1 and IRF4 in the nuclear extracts of cells treated for 2 days. β-Actin was measured as a loading control. The levels of IKZF1 and IRF4 relative to DMSO control are indicated under the blots and are relative to the DMSO treated control.
Figure 7.
Figure 7.
Effect of Pom on MHC-I, ICAM-1, and B7-2 surface expression in raji cells. (A-C) Raji cells were treated for two days with DMSO control or Pom (0, 1, or 10 µM). A representative surface expression histogram for (A) MHC-I, (B) ICAM-1, and (C) B7-2 is shown for DMSO (solid line), 1 µM Pom (dashed line), and 10 µM Pom (dotted line). The isotype control is shown in grey. (D) The average fold change in MHC-I, ICAM-1 and B7-2 expression. The data represents the averages ± standard deviations from three independent experiments. The asterisk indicates p < 0.05. (E) Nuclear and cytoplasmic extracts were prepared 2 days after treatment and analyzed for MHC-I (cytoplasmic) and IKZF1 and IRF4 (nuclear) expression. β-Actin was measured as a loading control. The levels of MHC-I, IKZF1 and IRF4 relative to the DMSO-treated control are indicated under the blots. Note the viability of Raji cells remained > 95% for all treatments.
Figure 8.
Figure 8.
Pom increases NK cell-mediated cytotoxicity against Daudi cells but not raji cells. Daudi cells (A) or Raji cells (B) were treated with DMSO control, 1 µM Pom or 10 µM Pom for 2 days and then assayed for NK cell-mediated cytotoxicity with YTS effector cells using effector-to-target ratios of 0.25:1 to 5:1. Shown are the average results ± standard deviation from 3 independent experiments for each cell line. The asterisk indicates p < 0.05 compared with DMSO control.
Figure 9.
Figure 9.
Pom increases T-cell activation by Daudi and raji cells. Daudi (A) or Raji (B) cells were treated with 0, 1, or 10 µM Pom for 2 days and co-incubated with Jurkat IL-2 reporter T-cells in the presence of various concentrations of anti-CD3 antibody. Luminescence, a measure of T cell activation, was measured after 6 hours. This experiment was performed three times. Data from one representative experiment for each cell line is shown in the left panel. The activation is expressed as relative luminescence unit (RLU) and plotted as a 4PL regression graph and the error bars represent standard deviations from technical replicates. Right panel shows average fold changes in T-cell activation by 1 and 10 µM Pom-treated Daudi cells (A) or Raji cells (B) relative to DMSO control in the presence of 0.16µM anti-CD3 antibody. Error bars represent standard deviations from three independent experiments. *p < 0.05, ***p < 0.005.
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Grants and funding

This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, and a CRADA between the NCI and Celgene Corporation.