Interferon-γ Secretion by t(9;22) Acute Lymphoblastic Leukemia-Derived Dendritic Cells

Abstract

Interferon (IFN)-γ plays an important role in immunity and anti-tumor activity. It is produced by lymphocytes, but was recently shown to be also produced by human myeloid dendritic cells (DCs). We have shown that human mature t(9;22) acute lymphoblastic leukemia-derived (ALL) DCs induced autologous cytotoxic T cell responses and therefore asked whether t(9;22) ALL-DC secreted IFN-γ. IFN-γ varied among three cell line-derived ALL-DCs; median production from seven patient ALL-DCs was 3450pg/ml (range 1450-8675). IFN-γ production was dependent on maturation of ALL-DCs. This is the first demonstration of IFN-γ production by t(9;22) ALL-DCs.

Introduction

Interferon (IFN)-γ is critical for immunity against pathogens and tumors. It is produced by natural killer and natural killer T cells as part of the innate immune response and by T_H1 CD4 and cytotoxic CD8 T cells as part of the adaptive immune response. IFN-γ secretion from these cells is mediated in part by IL-12 released from regulatory immune cells including dendritic cells (DC) and macrophages. Recently, IFN-γ was shown to be produced by murine myeloid, lymphoid and IFN-producing killer dendritic cells (IKDCs) (1). In humans, IFN-γ has only been found to be secreted by myeloid DCs. We have shown that human mature t(9;22) acute lymphoblastic leukemia-derived dendritic cells (ALL-DC) induced autologous cytotoxic T cell responses against unmodified blasts in a patient who achieved remission (2). This is achieved partly by up-regulation of two components of the antigen processing machinery (APM), HLA class I heavy chain antigen (HLA-HC) and tapasin, in these t(9;22) ALL-DC compared to the unmodified blasts (3). The cytokine profile secreted by these cells is largely unknown. We asked whether t(9;22) ALL-DC secreted IFN-γ to assist in leukemia cell killing via T cell stimulation, polarization or increased expression of the APM components.

Materials and Methods

Cell lines and patient samples

Human t(9;22) ALL cell lines were cultured as previously described (2). The K562 cell line was purchased from the American Type Culture Collection (Manassas, Virginia). Bone marrow samples from seven consecutive t(9;22) ALL patients (containing >85% blasts) and peripheral blood mononuclear cells from four normal volunteers were used after approval by the Institute’s Scientific Review Committee and Institutional Review Board.

Cytokines

Interleukin (IL)-1β, IL-3, IL-7, tumor necrosis factor (TNF)-α and stem cell factor (SCF) were purchased from R & D, Minneapolis, MN. Type B CpG oligodeoxynucleotide (ODN) 2006 was purchased from Coley Pharmaceutical Group, Kanata, Canada and CD40 ligand (L) was provided by Amgen, Thousand Oaks, CA.

Antibodies

Fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies (mAb) against CD40, CD54, CD80, CD86 and IFN-γ peridinin chlorophyll (PerCP)-conjugated mAb against CD8 and Phycoerythrin (PE)-conjugated mAb against IL-4 were from BD Pharmingen, San Diego, CA. The Allophycocyanin (APC)-conjugated mAb against CD3 was from Caltag (Burlingame, CA), Alexa-647-conjugated HLA-DR antibody was from BioLegend (San Diego, CA); anti-tapasin mAb TO-3 and the mAb HC-10 which recognizes a determinant expressed on all β₂m free HLA-B and C heavy chains and on β₂m free HLA-A10, -A28, -29, -A30, -A31, -A32, and -A33 heavy chains (4) were developed and characterised as described (5). PE goat anti-mouse IgG (Caltag) was used as a secondary antibody. The neutralizing goat anti-human IFN-γ antibody and goat IgG isotype were purchased from Sigma (St. Louis, MO).

Generation of ALL-DC

ALL-DC were generated as previously described (2, 3).

Cytokine concentrations in conditioned media

Supernatants from triplicate day eight cultures were tested for IL-12 and IFN-γ using enzyme-linked immunosorbent assay, (R & D).

Flow cytometric analysis

Analysis of cell surface and intracellularly stained cells was performed as previously described (2, 3).

Tapasin and HLA-HC detection

Intracellular analysis of proteins was performed as previously described (3).

Allogeneic-Mixed Lymphocyte Reaction (allo-MLR)

Allo-MLR was conducted as previously described (2).

T Cell Polarization

Multiparameter flow cytometry was used to detect intracellular levels of IFN-γ and IL-4 simultaneously with the expression of CD3 and CD8. ALL-DC were treated with medium, IgG isotype control, or IFN-γ neutralizing antibody at the ND₅₀ and 100x the ND₅₀ and co-cultured with stimulated allogeneic T cells for 48hrs at 37°C. Cells were stained and analyzed by flow cytometry as described (6).

Statistical analysis

Statistical analysis was performed by the Student’s paired two-tailed t test using Microsoft Excel.

Results and Discussion

We recently demonstrated that ALL-DC undergo differentiation in two steps (2); the first step generates immature ALL-DC with low stimulatory potential and the second step generates mature, stimulatory ALL-DC. We studied cytokine secretion from these ALL-DCs to determine if IFN-γ aided in leukemia cell killing. As expected, immature ALL-DC secreted neither IL-12 nor IFN-γ. Unexpectedly, mature ALL-DC did not secrete IL-12, however, mature ALL-DC derived from one t(9;22) ALL cell line, Z181, and from primary blasts from seven t(9;22) ALL patients, secreted IFN-γ after treatment with the maturating agents CD40L and TNF-α (Figure 1). These data suggested that mature ALL-DC could produce IFN-γ independent of IL-12.

Figure 1. IFN-γ secretion from t(9;22) ALL-DC.

IFN-γ secretion was measured from ALL-DC generated from three cell lines and seven patient samples on day 8 of the standard cytokine culture.

Interestingly, DCs generated from the other cell lines, OM9;22 and Z119 did not secrete significant amounts of IFN-γ even though these cells were determined to be mature by CD80+/CD86+ expression following culture with CD40L and TNF-α. We hypothesized that CD40L and TNF-α did not induce full maturation of the ALL-DCs generated from OM9;22 cells, and that further maturation was needed. Following the addition of CD40L, TNF-α and CpG ODN, IFN-γ (109.3 ± 20.23 pg/ml) was secreted from OM9;22-derived DCs (Figure 2).

Figure 2. IFN-γ secretion from immature and mature OM9;22 AML-DCs.

(A) IFN-γ secretion was measured by ELISA and (B) immunophenotype was measured by flow cytometry from immature OM9;22 ALL-DC treated with different maturating agent combinations.

We next asked what role IFN-γ plays in this system. We tested three known mechanisms: T cell stimulation, T cell polarization and intracellular expression of the APM components tapasin and HLA-HC. We measured T cell stimulation by ALL-DC generated from three cell lines, Z119 (no IFN-γ secretion), Z181 (high IFN-γ secretion) and OM9;22 (IFN-γ secretion following exposure to CpG ODN), and two patient samples. There were no statistically significant changes in MLR responses after the addition of IFN-γ neutralizing antibodies. Subsequently, intracellular expression of tapasin and HLA-HC was tested in ALL-DC generated from Z119 (no IFN-γ secretion) and Z181 (high IFN-γ secretion) treated with neutralizing IFN-γ antibody; expression remained unchanged. Finally, no effect of IFN-γ neutralizing antibody was detected on T cell polarization by ALL-DC generated from Z181. To demonstrate that IFN-γ was functional, supernatants from Z119 and Z181 ALL-DC were added to the cell line K562, previously shown to up-regulate HLA-DR in response to IFN-γ exposure (7). Supernatants from Z181 but not Z119 ALL-DC induced increased expression of HLA-DR (Figure 3). These data suggest that IFN-γ is functional, but the role of IFN-γ production by ALL-DC is still unknown.

Figure 3. IFN-γ induces up-regulation of HLA-DR in the K562 cell line.

HLA-DR expression was measured by flow cytometry in the K562 cell line after exposure to supernatants from ALL-DC generated from the Z119 (solid) or Z181 (gray) cell lines.

In summary, we demonstrate for the first time that t(9;22) ALL-DCs secrete IFN-γ independent of IL-12. Though our studies did not elucidate the exact role of IFN-γ, we propose that similar to murine IKDCs (8), IFN-γ may enhance the ability of t(9;22) ALL-DC to induce direct or indirect cytotoxicity against unmodified blasts. These cells may be useful in the development of immunotherapeutic approaches for the treatment of t(9;22) ALL.