The immunomodulatory-drug, lenalidomide, sustains and enhances interferon-α production by human plasmacytoid dendritic cells

Abstract

Background: Lenalidomide (LEN), an immunomodulatory drug (IMiD), is currently used for treatment of multiple myeloma (MM). LEN potentiates T cell and natural killer cell functions. However, the cellular and molecular mechanisms underlying the immunomodulatory effects of LEN remain unclear. We focused on the effects of LEN on human plasmacytoid dendritic cells (pDCs), which are the major source of interferon (IFN)-α in the blood and play a central role in innate immune responses. Results: We found that bortezomib, a proteasome inhibitor used to treat MM, killed pDCs but that 0.1-3 μM LEN (covering clinical plasma concentration range) did not affect pDC survival or CD86 expression. Bortezomib inhibited pDC-derived IFN-α production in a dose-dependent fashion, but 0.1-3 µM LEN sustained pDC-derived IFN-α production when stimulated with an optimal concentration of CpG-ODN 2216 (3 μM). In pDCs stimulated with a low concentration of CpG-ODN (0.1 μM), LEN enhanced IFN-α production. These results indicated that LEN, when used at a clinically relevant concentration, can potentially enhance IFN-α production by pDCs. Conclusion: Collectively, our findings unveiled a novel target of LEN and extend the repertoire of the drug's known immunomodulatory effects. These effects may explain the low incidence of herpes zoster viral infection observed during LEN treatment compared with bortezomib treatment. LEN may function as an IMiD affecting a wide array of immune cells, including pDCs, leading to amplification of a positive immune axis able to eliminate MM cells.

Keywords: IMiDs; lenalidomide; multiple myeloma; plasmacyotid DCs; type I IFNs.

Figure 1

Effects of lenalidomide on plasmacytoid dendritic cell survival. Human plasmacytoid dendritic cells (pDCs) were incubated for 24 h with the indicated concentrations of lenalidomide (LEN), bortezomib, or vehicle in the presence of 3 μM CpG-ODN 2216. After 24 h, viable cells were measured by annexin V staining and flow cytometry. Percentages of annexin V-positive cells are indicated. Data are shown as means ± SEMs of six independent donors. Statistical significance was determined using paired Student’s t-tests (*=p<0.05, **=p<0.01; p-values were calculated for each concentration of LEN or bortezomib versus vehicle control).

Figure 2

Effects of lenalidomide on plasmacytoid dendritic cell maturation. Human plasmacytoid dendritic cells (pDCs) were incubated for 24 h with the indicated concentrations of lenalidomide (LEN), bortezomib (BOR), or vehicle in the presence of 3 μM CpG-ODN 2216. (A) After 24 h, CD86 expression on pDCs was analyzed using flow cytometry. Data were quantified as the mean fluorescence intensity (MFI), which was calculated by subtraction of the MFI for the isotype control from the MFI for cells stained with CD86 mAb. Data are shown as means ± SEMs of six independent donors. (B) CD4⁺ T cells were stimulated for 7 days with allogeneic pDCs pretreated with CpG, CpG + 1 μM LEN, or CpG + 30 nM bortezomib for 24 h. After culture, cells were quantitated by trypan blue dye-exclusion. Data are shown from four independent sets of experiments. Statistical significance was determined using paired Student’s t-tests (*=p<0.05, **=p<0.01; p-values were calculated for each concentration of LEN or bortezomib versus vehicle control). Abbreviation: N.E., not evaluated.

Figure 3

Frequency of blood plasmacytoid dendritic cells in multiple myeloma patients before and after treatment with lenalidomide. Frequency of plasmacytoid dendritic cells (pDCs) was calculated amongst total peripheral blood mononuclear cells (PBMCs) (at least 2×10⁶ cells) from peripheral blood using flow cytometry. pDCs were detected as BDCA-2-positive population in the lineage (CD3, CD14, CD15, CD16, CD19, and CD56)-negative, CD11c-negative, and CD4-positive fraction. (A) A representative flow cytometry analysis showing pDCs in PBMCs from six newly-diagnosed multiple myeloma (MM) patients before treatment. Numbers indicate percentages of the gated fraction amongst total PBMCs. (B) Each dot shown is derived from 10 healthy donors and six newly-diagnosed MM patients before and after one cycle of lenalidomide (LEN)-dexamethasone administration. Statistical significance was determined using unpaired Student’s t-tests of healthy donors versus MM patients before LEN treatment, or paired Student’s t-tests of MM patients before LEN treatment versus MM patients after LEN treatment.

Figure 4

Clinical concentrations of lenalidomide (LEN) sustained IFN-α production by peripheral blood mononuclear cells (PBMCs). Human PBMCs were incubated for 24 h with the indicated concentrations of LEN or vehicle in the presence of 3 μM CpG-ODN 2216. After 24 h, the concentrations of IFN-α in the culture supernatants were measured by ELISA. Data are shown as means ± SEMs of seven independent donors. The data were normalized to the value obtained for the vehicle control. The mean (range) of absolute concentrations of CpG-ODN 2216 + vehicle control was 6396 pg/mL (1006–12,938 pg/mL). Statistical significance was determined using paired Student’s t-tests; p-values were calculated for each concentration of LEN versus vehicle control.

Figure 5

Clinical concentrations of lenalidomide (LEN) sustained and potentially enhanced IFN-α production by human plasmacytoid dendritic cells. (A,B) Purified plasmacytoid dendritic cells (pDCs) were incubated for 24 h with the indicated concentrations of bortezomib, LEN, or vehicle in the presence of 3 µM CpG-ODN 2216. Data are shown as means ± SEMs of five independent donors for bortezomib experiments (A) and 12 independent donors for LEN experiments (B). The data were normalized to the value obtained from cells treated with vehicle control. The mean (range) of absolute concentrations of CpG-ODN 2216 + vehicle control was 29,786 pg/mL (12,921–56,217 pg/mL). (C) Purified pDCs were incubated for 24 h with 1 μM LEN or vehicle in the presence of the indicated concentrations of CpG-ODN 2216. Data are derived from at least six independent donors at each CpG-ODN 2216 concentration and are shown as means ± SEMs. The concentrations of IFN-α in culture supernatants were measured by ELISA. Statistical significance was determined using paired Student’s t-tests (*=p<0.05, **=p<0.01; p-values were calculated for each concentration of LEN or bortezomib versus vehicle control).