Optimizing immunotherapy in multiple myeloma: Restoring the function of patients' monocyte-derived dendritic cells by inhibiting p38 or activating MEK/ERK MAPK and neutralizing interleukin-6 in progenitor cells

Abstract

Previous studies demonstrated that circulating dendritic cells (DCs) in myeloma patients were functionally abnormal. However, the phenotype and function of patients' monocyte-derived DCs (MoDCs), which are commonly used for immunotherapy, were poorly defined. This study was undertaken to examine the quality of MoDCs from myeloma patients compared with cells from healthy donors. We found that patient-derived MoDCs are phenotypically and functionally defective. Compared with their normal counterparts, patient-derived, mature MoDCs expressed significantly lower levels of CD1a, CD40, CD80, and HLA-DR and were poor at activating alloreactive T cells, presenting recall antigen, and activating autologous antigen- and myeloma-specific T cells. These abnormalities may be attributed to elevated production of autocrine cytokines such as IL-6, activated p38 and STAT3, and inhibited MEK/ERK signaling pathways in the progenitor cells. Treatment with neutralizing IL-6-specific antibody and, more importantly, p38 inhibitor, or both, could correct these abnormalities. Treating patient-derived cells with these agents not only significantly increased cell yield but also produced MoDCs that were as functional as their normal counterparts. Thus, this study has delineated the mechanistic defects of MoDCs from myeloma patients and identified ways for restoring the function of the cells to improve the efficacy of DC-based immunotherapy in this disease.

Figure 1.

Phenotypic and functional properties of MoDCs from healthy donors (HD) and myeloma patients (MM). Representative histograms showing the expression of CD1a, CD40, CD80, and HLA-DR on (A) immature and (B) mature MoDCs generated from a healthy donor and a myeloma patient. Numbers inside the histograms represent the mean fluorescence intensity (MFI). (C) Pooled data depicting the expression (MFI) of CD1a, CD40, CD80, and HLA-DR on mature MoDCs from all tested healthy donors (n = 10) and myeloma patients (n = 12). Bars indicate the mean MFI. (D) Allostimulatory capacity of mature MoDCs in activating alloreactive T cells in an MLR assay. Shown are the mean ± SD of the results obtained with cells from 7 healthy donors and 7 myeloma patients. (E) Antigen-presentation capacity of MoDCs to autologous T cells. Shown are the mean ± SD of the results of cells presenting recalled antigen PPD to autologous T cells obtained from 5 healthy donors and 5 myeloma patients. In these experiments, different T cell/DC ratios (10:1, 100:1, and 1000:1; noted in the figure) were used. * P values less than .05.

Figure 2.

Expression and production of cytokines by DC progenitor cells from healthy donors (HD) and myeloma patients (MM). (A) RT-PCR detecting mRNA expression for IL-6, IL-10, and TGF-β1 in freshly isolated monocytes (day 0) and day-2–cultured cells from a healthy donor and a myeloma patient. Representative results of 4 independent experiments are shown. ELISA detecting (B) IL-6 or (C) TGF-β1 in the supernatants of day-3 cultures of the cells. Shown are the mean ± SD of the results obtained from all tested healthy donors (n = 7 for IL-6, and n = 3 for TGF-β1) and myeloma patients (n = 7 for IL-6, and n = 3 for TGF-β1). ** P values less than .01.

Figure 3.

Effects of cytokine-neutralizing antibodies and p38 inhibitor on the phenotype of MoDCs from myeloma patients. (A) Representative histograms showing the expression of CD1a, CD40, CD80, HLA-ABC, and HLA-DR on patient-derived mature MoDCs generated in cultures with or without addition, individually or in a combination (Ab comb), of all 3 neutralizing antibodies (Ab; 10 μg/mL) to IL-6, IL-10, or TGF-β1; p38 inhibitor; or p38 inhibitor and IL-6 antibody. Numbers inside the histograms represent the MFI. (B) Pooled data depicting the expression (MFI) of CD1a, CD40, CD80, HLA-ABC, and HLA-DR on the cells from all tested myeloma patients (n = 6) treated with IL-6 antibody, p38 inhibitor, or both. Bars indicate the mean MFI. *P values less than .05; **P values less than .01.

Figure 4.

Effects of cytokine-neutralizing antibodies and p38 inhibitor on functional properties and yield of MoDCs from myeloma patients. (A) Allostimulatory and (B) antigen-presentation capacities of myeloma patient–derived MoDCs generated in cultures treated with IL-6 antibody, p38 inhibitor, or both. Shown are the mean ± SD of results obtained with the cells from all tested patients (n = 6 for MLR, and n = 4 for PPD); a T/MoDC ratio of 100:1 was used. (C) Cytotoxicity of myeloma-specific CTLs induced by myeloma lysate–pulsed autologous MoDCs or p38 inhibitor–treated MoDCs from 2 myeloma patients (MM1 and MM2). Target cells were lysate-pulsed autologous MoDCs. Unpulsed MoDCs were used as controls. (D) Cell yields determined by microscopic cell count of large, DC-like cells in cultures treated with IL-6 antibody, p38 inhibitor, or both. Shown are the mean ± SD of the results obtained with the cells from 4 patients. In panels A, B, and D, pooled data of normal MoDCs from healthy donors (n = 7 for MLR, n = 5 for PPD, and n = 4 for cell yields) were included for comparison (▪). *P values less than .05; **P values less than .01.

Figure 5.

Effects of p38 inhibitor on cytokine expression and production by myeloma patient–derived MoDC progenitor cells. (A) RT-PCR detecting mRNA expression for IL-6, IL-10, and TGF-β1 in day-2–cultured cells from a myeloma patient. Representative results of 4 independent experiments are shown. ELISA detecting (B) IL-6 or (C) TGF-β1 in the supernatants of day-3 cultures of the cells. Shown are the mean ± SD of the results obtained from tested myeloma patients (n = 5 for IL-6, and n = 3 for TGF-β1). *P values less than .05.

Figure 6.

Signaling pathways in the differentiating MoDCs obtained from healthy donors or myeloma patients. (A) Western-blot analysis showing protein levels of phosphorylated (p) and nonphosphorylated p38, MEK1/2, ERK1/2, and STAT3 in freshly isolated monocytes (day 0) and day-2–cultured cells from a healthy donor and a myeloma patient. (B) Effects of IL-6–neutralizing antibody (Ab), p38 inhibitor (P38I), or both on signaling pathways in myeloma patient–derived cells. (C) Effects of TCCM and p38 inhibitor on signaling pathways in normal MoDC progenitor cells from healthy donors. In all the experiments, day-2–cultured cells were examined. Representative results of 3 independent experiments are shown.