Plasmacytoid Dendritic Cells in Patients with MGUS and Multiple Myeloma

Abstract

Background: Plasmacytoid dendritic cells (pDCs) play prominent roles in mediating innate and adaptive immune responses. However, it is unclear how pDCs contribute to the immunosuppressive tumor microenvironment described in multiple myeloma (MM).

Methods: Newly diagnosed myeloma patients (MM, n = 37) were analyzed to determine the pDC counts in comparison to peripheral blood (PB, n = 53) and bone marrow (BM, n = 10) samples of age-matched healthy donors (HD) using flow cytometry. Second, proliferation of myeloma tumor cells in the presence of freshly isolated pDCs was examined. Third, production of IFNα by pDCs co-cultured with MM cells was determined by intracellular staining.

Results: We found a highly significant reduction of circulating pDCs (p < 0.0001) and in bone marrow (p < 0.0001) of MM patients compared to HD. We also observed a significant decrease of pDCs (p = 0.004) in BM in patients with monoclonal gammopathy of undetermined significance (MGUS, n = 12). Importantly, we determined that pDCs promote proliferation specifically of MM cells and not the stromal cells and that pDCs secrete IFNα upon co-culture with MM tumor cells.

Conclusions: Our results show altered pDC frequencies in the BM microenvironment in MGUS and MM patients at diagnosis. We showed the tumor-promoting function of pDCs that may mediate immune deficiencies affecting long-term disease control and treatment outcome.

Keywords: MGUS; immunosuppressive tumor microenvironment; multiple myeloma; plasmacytoid dendritic cells.

Figure 1

Plasmacytoid dendritic cells in healthy donors and multiple myeloma patients: (a) PDCs were analyzed by flow cytometry as CD123, CD303, CD304, and CD45 positive cells. Representative FACS analysis showing a pDC population in a MM patient; (b) PDC frequencies were analyzed in peripheral blood (PB, n = 53) and bone marrow (BM, n = 10) samples of healthy donors (HD) and compared to newly diagnosed myeloma patients (MM, n = 37). Each box shows the median with min and max values. Significant reductions of pDCs were determined as *** p < 0.0001 using the non-parametric Mann–Whitney test.

Figure 2

Bone marrow pDCs in MGUS and MM patients. PDC frequencies were analyzed in bone marrow (BM) in healthy donors (HD), MGUS and myeloma patients (MM). Each box shows the median with min and max values. Significant reductions of pDCs were determined as *** p < 0.0001 and ** p = 0.004 using the non-parametric Mann–Whitney test.

Figure 3

Expansion of myeloma cells in the presence of pDCs: (a) Proliferation of MM cell lines including U266 (5 × 10⁵ cells/1 mL); (b) KMS-11 (5 × 10⁵ cells/1 mL) alone and in the presence of freshly sorted pDCs (10⁵ cells/1 mL) at a 5:1 ratio for 24 h (pale grey) and 72 h (dark grey) co-cultured in 24-well plates; (c) Proliferation of MM cell lines including U266 (5 × 10⁵ cells/1 mL); (d) KMS-11 (5 × 10⁵ cells/1 mL) alone and in the presence of the HS-5 cell line (10⁵ cells/1 mL) at a 5:1 ratio for 24 h (pale grey) and 72 h (dark grey) co-cultured in 24-well plates. Data are shown as mean ± SD from three independent experiments. Significant differences in pDC co-cultures were determined using the unpaired t test as **** p < 0.0001 (U266) and ** p = 0.002 and p = 0.007 (KMS-11) respectively.

Figure 3

Expansion of myeloma cells in the presence of pDCs: (a) Proliferation of MM cell lines including U266 (5 × 10⁵ cells/1 mL); (b) KMS-11 (5 × 10⁵ cells/1 mL) alone and in the presence of freshly sorted pDCs (10⁵ cells/1 mL) at a 5:1 ratio for 24 h (pale grey) and 72 h (dark grey) co-cultured in 24-well plates; (c) Proliferation of MM cell lines including U266 (5 × 10⁵ cells/1 mL); (d) KMS-11 (5 × 10⁵ cells/1 mL) alone and in the presence of the HS-5 cell line (10⁵ cells/1 mL) at a 5:1 ratio for 24 h (pale grey) and 72 h (dark grey) co-cultured in 24-well plates. Data are shown as mean ± SD from three independent experiments. Significant differences in pDC co-cultures were determined using the unpaired t test as **** p < 0.0001 (U266) and ** p = 0.002 and p = 0.007 (KMS-11) respectively.

Figure 4

The IFNα secretion by pDCs in the co-cultures with MM cells: (a) Proliferation of MM cell lines including U266, KMS-11, OPM-2 and LP-1 (5 × 10⁵ cells/1 mL) alone (white bars) and in the presence of freshly sorted pDCs (10⁵ cells/1 mL) at a 5:1 ratio for 24 h (grey bars) co-cultured in 24-well plates was analyzed. Numbers of IFNα producing pDCs are shown (black bars). Stimulation of pDCs alone with PMA and CI is shown (pale grey bars). Data are shown as mean ± SD from three independent experiments. Significant differences in pDC co-cultures were determined using the unpaired t test as * p < 0.01 (KMS-11, LP-1), ** p = 0.001 (U266) and *** p = 0.0005 (OPM-2) respectively. (b) Representative FACS plot analysis of pDC populations identified as CD123 and CD304 by surface staining. (c) Intracelllular staining showing pDC positive populations secreting IFNα.

Figure 4

The IFNα secretion by pDCs in the co-cultures with MM cells: (a) Proliferation of MM cell lines including U266, KMS-11, OPM-2 and LP-1 (5 × 10⁵ cells/1 mL) alone (white bars) and in the presence of freshly sorted pDCs (10⁵ cells/1 mL) at a 5:1 ratio for 24 h (grey bars) co-cultured in 24-well plates was analyzed. Numbers of IFNα producing pDCs are shown (black bars). Stimulation of pDCs alone with PMA and CI is shown (pale grey bars). Data are shown as mean ± SD from three independent experiments. Significant differences in pDC co-cultures were determined using the unpaired t test as * p < 0.01 (KMS-11, LP-1), ** p = 0.001 (U266) and *** p = 0.0005 (OPM-2) respectively. (b) Representative FACS plot analysis of pDC populations identified as CD123 and CD304 by surface staining. (c) Intracelllular staining showing pDC positive populations secreting IFNα.