Deregulated cytokine network and defective Th1 immune response in multiple myeloma

Abstract

Intracellular cytokine production by peripheral blood mononuclear cells (PBMC) was analysed in 51 patients with multiple myeloma (MM), 22 with monoclonal gammopathy of undetermined significance (MGUS) and 20 healthy subjects, as a parameter of immunological dysfunction in MM. An increased proportion of T cells and HLA-DR+ cells producing IL-6 was observed in MM patients with active disease (at diagnosis and relapsing) compared with patients in remission and with MGUS, whereas no difference of IFN-gamma+, IL-2+ PBMC between patients and controls was evident. Determination of serum cytokine levels demonstrated that the imbalanced IL-6 production by T cells and the defective anti-tumour Th1 cell activity were related to elevated levels of IL-6 and IL-12. In vitro studies of PHA- and anti-CD3/anti-CD28 MoAbs stimulation of PBMC demonstrated the ability of lymphocytes from MM patients to differentiate towards the Th1 subset in the presence of rIL-12. By contrast, addition of exogenous rIL-6 impaired IFN-gamma production by rIL-12-prompted T cells. Inhibition of Th1 polarization of the immune response by IL-6 was direct on T cells and not mediated by dendritic cells (DC). Evaluation of the ability of MM-derived DC to stimulate cell proliferation of allogenic T lymphocytes and produce IL-12 in vitro, in fact, suggested that MM-derived DC were functionally active. Taken as a whole, these results indicate that a deregulated cytokine network occurs in active MM. They also suggest that increased IL-6 production by peripheral T lymphocytes contributes to the immune dysfunction observed in MM, and enables tumour cells to escape immune surveillance by preventing the anti-tumour Th1 immune response.

Fig. 1

Intracellular cytokine production by peripheral blood mononuclear cells (PBMC) in MM and MGUS patients and healthy subjects. Purified PBMC were stimulated with phorbol myristate acetate/ionomycin or with LPS for 5 h, then analysed for intracellular cytokine synthesis. Patients with active MM (at diagnosis and in relapsing disease) showed significantly higher percentage values of IL-6-producing PBMC compared with patients in remission, patients with MGUS and healthy subjects. No difference in Th1-type lymphocytes was observed. () IFN-γ⁺; (□) IL-2⁺; () IL-4⁺; (▪) IL-6⁺. *P < 0·005.

Fig. 2

Representative double-fluorescence staining of peripheral CD3⁺/IL-6⁺ and HLA-DR⁺/IL-6⁺ lymphocytes in two MM patients at different clinical stages, in one MGUS patient and in a healthy subject. An expansion of IL-6-producing HLA-DR⁺ lymphocytes was observed in the patient with relapsing MM (a, b) compared with the patient in remission MM (c, d), the MGUS patient (e, f) and the control (g, h).

Fig. 3

Effect of rIL-6 and rIL-12 on IFN-γ production by stimulated patients' lymphocytes. PBMC (1 × 10⁶) purified from 32 MM patients at different clinical stages were stimulated in vitro with PHA (5 µg/ml) or with immobilized anti-CD3 MoAb (1 µg/ml) and soluble anti-CD28 MoAb (1 µg/ml) in the presence of culture medium (–), rIL-12 (400 ng/ml) or rIL-6 (100 U/ml). After 5 days, IFN-γ production was evaluated by flow cytometry. Addition of rIL-12 induced a Th1 polarization of cultured PBMC that was significantly inhibited by the presence of rIL-6.

Fig. 4

Functional studies of monocytes-derived dendritic cells (DC). DC were generated from adherent PBMC cultured with GM-CSF (800 U/ml) and rIL-4 (400 U/ml) and stimulated with LPS (1 ng/ml) for the final 24 h of culture. (a) T cell stimulatory function (MLR assay) of patient- (•) and healthy subject-derived (▴) DC. LPS-stimulated DC from 15 MM patients, 11 MGUS patients and seven healthy subjects were treated with mitomycin C and used at different concentrations. Responder cells (1 × 10⁵ per well) were obtained from an allogeneic donor. No difference in allostimulatory activity between patient- and control-derived DC was evident. (b) Supernatant fluids of cultured DC generated from patients and healthy subjects were collected and IL-12 content was evaluated by ELISA. Patient-derived DC released large amounts of this cytokine, comparable with control-derived DC.