Quantitative and Functional Alterations of 6-Sulfo Lacnac Dendritic Cells in Multiple Myeloma

Multiple myeloma (MM) is characterized by expansion of abnormal plasma cells in the bone marrow (BM). Monocytes and macrophages have been shown to accumulate in the microenvironment / BM of MM patients while promoting MM cells survival and proliferation. Recently, another population of myeloid cells expressing the M-DC8 antigen containing a 6-sulfo LacNAc structure, called 6-sulfo LacNac dendritic cells (Slan-DCs) has been described. Functionally, Slan-DCs have potent pro-inflammatory properties based on their capacity to produce large amounts of Tumor Necrosis Factor-α and Interleukin (IL) 12p70 upon stimulation with Toll-like receptor ligands such as R848. They also express C-X-C chemokine receptor type 4 which is important in homing to the BM. Nevertheless, little is known about microenvironmental interactions between MM cells and Slan-DCs.

To gain insights on the Slan-DCs' potential role in MM pathophysiology, we evaluated their phenotype and functions in the BM and blood of patients with newly diagnosed symptomatic MM. Using multicolor flow cytometry, we performed quantitative and functional analyses. Compared to healthy donors (n=11), the percentages and absolute numbers of circulating HLA-DR⁺ M-DC8⁺ Slan-DCs were significantly decreased in the blood of MM patients (n=23) (for absolute number: 20.64 vs 7.61 Slan-DCs/μL, p=0.0015 and for percentage: 1.06 vs 0.49%, p=0.029). In contrast, there were no significant difference in the frequencies of Slan-DCs in the patients BM.

We next addressed their functional properties, in particular cytokine production. We observed a significant decrease in IL-12-producing Slan-DCs from MM patients as compared to healthy donors both in blood (25.88% versus 56.86% respectively, p<0.0001) and BM (25.11% versus 48.83% respectively, p=0.016). In order to investigate whether the decrease in IL-12 secretion was due to the MM cells, circulating Slan-DCs from healthy controls were stimulated with R848 in the presence of 4 different MM cell lines. The capacity of Slan-DCs from healthy controls to secrete IL-12 under this stimulation was measured by ELISA. We observed that the RPMI-8226, KMS-12-PE and LP-1 MM cell lines inhibited R848-induced IL-12 secretion by Slan-DCs. Furthermore, we could show that primary MM cells from patients at diagnosis (magnetically sorted CD138+ cells from patients) were able to inhibit IL-12 secretion by Slan-DCs from healthy donors (n=5).

Since MM cells inhibited Slan-DCs secretion of IL-12 and given the pivotal role of IL-12 in Th1 polarization, we investigated whether MM cells could modulate the capacity of Slan-DCs to induce T cell proliferation and Th1 differentiation. To this aim, we co-cultured T cells and Slan-DCs (n=5). Here, we found that normal Slan-DCs can efficiently induce the proliferation of T cells. In addition, their ability to promote T cell proliferation was more pronounced after R848 priming. However, after co-culture with RPMI-8226 MM cell line, the fraction of proliferating T cells at day 7 was significantly decreased (58.14% versus 28.37% ; p=0.03). Moreover, RPMI-8226 MM cell line markedly impaired the capacity of Slan-DCs to induce the differentiation of naïve CD4+ T cells into IFNγ-producing Th1 cells (34.59% versus 7.89% ; p=0.04).

Based on the above results, we hypothesized that Slan-DCs' pro-inflammatory phenotype could also be impacted by MM cells. Thus, freshly-sorted Slan-DCs were cultured with or without MM cell lines and their phenotype was analyzed by flow cytometry (n=9). When Slan-DCs were cultured in the presence of MM cells, expression of CD14 and CD16, but not CD11b, was induced, in line with a phenotype of an "intermediate" monocytes population. Moreover, in the presence of MM cells, the expression of the costimulatory molecule CD86 was weakly induced on Slan-DCs (Mean of Fluorescence Intensity = 102049 versus 199470, p=0.004).

In conclusion, these results provide evidence that MM cells are able to significantly impair the capacity of Slan-DCs to secrete IL-12 and stimulate CD3+ T cell proliferation and to polarize naïve CD4+ T cell into Th1 cells. The latter suggests that MM cells are able to strongly suppress the pro-inflammatory function of Slan-DCs, directing them towards a more tolerogenic profile. These findings reveal a novel mechanism of immune escape in MM, and may pave the way for newer immune-intervention strategies in combination with other currently available therapies.