Multiple Myeloma Generates Regulatory T-Cells in a Contact-Dependent Manner Independent of TGF-Beta and IL-10.

We have previously shown that Regulatory T-cells (T_Reg cells) are increased in the peripheral blood (PB) and bone marrow (BM) of patients with multiple myeloma (MM), related to the stage of disease. Therefore, to investigate whether the tumour cells in MM generate and/or expand T_Reg cells as a method of immuno-surveillance avoidance, we developed an in vitro model of T_Reg cell generation.

PBMCs from PB of healthy donors were co-cultured with human myeloma cell line, U266B, for 7 days. Using a sequential gating strategy, T_Reg cells identified as CD4⁺/CD25⁺/FoxP3⁺ T-cells were expressed as a percentage of the CD4⁺ T-cell population. Experiments were repeated after CD25 depletion ± CD4 selection and using transwells to examine cell contact dependence. The tumour generated T_Reg cells (tT_Reg cells) were isolated on day 7 by FACS and compared with naturally occurring T_Reg cells (nT_Reg cells).

Co-culture of unselected PBMC's demonstrated a non-significant increase in T_Reg cells compared with controls (n=8; controls 3.8±0.8%, co-culture 6.7±1%, p=0.06). However, co-culture of CD4⁺CD25⁻ T-cells with U266 resulted in a significant increase in tT_Reg cells (n=15; control 0.6±0.3%; co-culture 31.5±3.6%, p<0.0001). tT_Reg cell generation was abrogated when contact was prevented (n=7; control 0.17±0.05%; co-culture 30.6±4.9%; addition of transwell 0.1±0.04%, p=0.009). TCR analysis by PCR on tT_Reg cells revealed a polyclonal nature of those cells. Blocking experiments with anti-IL-10, anti-ICOS-L & anti-TGFb, in addition to the TGFb-specific antagonist, LAP, did not show any reduction in tT_Reg cell generation. tT_Reg cells demonstrated full suppressive capabilities in a functional suppression assay. Phenotypically, tT_Reg cells demonstrated altered expression of key proteins: FoxP3 MFI was significantly lower (925 ± 47 vs. 1960 ± 109 in nT_Reg cells, p=0.0001), GITR expression was significantly higher (70±4% vs. 10±2.8%, p<0.0001) and PD1 expression was significantly higher (50±9% vs. 5.2±0.8%, p=0.003). CD-62L expression was lower in tT_Reg cells (88±2% vs. 97±0.5%, p=0.007). When tT_Reg cells were isolated by FACS on day 7 and stimulated for a further 5 days with IL-2 and CD3/CD28 coated beads, differentiation to FoxP⁺/IL-17⁺ and FoxP3⁻/IL-17⁺ CD4⁺ cells was demonstrated with similar efficiency to nT_Reg cells (2.6±1.8% vs 3.6±2.4% p=0.7 and 4.6±4.0% vs 4.5±3.8% p=0.8, respectively). The addition of U266 pre-treated with thalidomide, lenalidomide and an HDAC inhibitor for 24 hours prior to co-culture demonstrated that thalidomide and HDAC inhibition significantly reduced the generation even at a low concentrations (42.6±24.5% without vs. 20.7±11.9% with thalidomide, p=0.009 and 9.8±3.3% with HDAC inhibition, p=0.003), whereas the addition of lenalidomide did not influence tT_Reg cell generation.

The tumour cells of MM directly generate fully functional T_Reg cells in a contact dependent manner independent of known inducing cytokines and ligands. This is an active process under epigenetic control. The exact mechanism of induction is under investigation.

No relevant conflicts of interest to declare.