IMiDs activate effector immune cells and reduce inhibitory immune cells in MM in vitro. PBMCs from patients with MM (MM-PBMC) were stimulated with anti-CD3 Ab and incubated for 6 days in the absence or presence of lenalidomide and pomalidomide. IMiDs effect on effector cell activation was analyzed using flow cytometry and cytokine protein array. (A) Histogram plots by flow cytometric analysis show membrane-expression of positive-costimulatory signaling molecules CD28, ICOS, and ICOSL on gated MM-PBMC CD3⁺ cells incubated for 3 days in the absence or presence of lenalidomide and pomalidomide. Shadowed profiles indicate isotype-matched control immunoglobulin (Ig) staining. One representative of 3 independent experiments is shown. Statistical significance indicated (t test, 1-tailed distribution, P < .05). (B) IMiDs induced proliferation of effector cells in MM-PBMCs is shown by CFSE-staining flow cytometric analysis. Proliferating effector cells were identified by CFSE costaining in gated CD4 PECy5 (CD4T cells), CD8 PECy7 (CD8 T cells), or CD56 PE/CD8 PECy7 (NKT cells) positive subpopulations. Proliferation is represented by division index of each cell population. One representative of 3 independent experiments is shown. Statistical significance indicated (t test, 1-tailed distribution, P < .05). (C) IMiDs effect on cytokine production was determined in MM-PBMC effector cells by intracytoplasmic cytokine staining flow cytometric analysis. MM-PBMCs were stimulated with anti-CD3 Ab and cultured for 16 hours with or without lenalidomide and pomalidomide, and intracytoplasmic expression (percent positive stained cells) of IL-2 (PE) was shown in gated effector cell subpopulations, as shown in the figure by side scatter dot plots. y-axis represents IL-2 PE staining and x-axis represents CD4T cells (top panel), CD8 T cells (middle panel), and NKT cells (bottom panel). One representative of 3 independent experiments is shown. Statistical significance indicated (t test, 1-tailed distribution, P < .05). (D) Secreted IFNγ, IL-2, and IL-6 were measured by cytokine protein array in the supernatants of anti-CD3 Ab-stimulated MM-BMMCs in the absence or presence of lenalidomide and pomalidomide for 24 hours. Expression of cytokines was determined using ImageJ 1.37v (http://rsb.info.nih.gov/ij/) densitometric analysis. Average and SD of triplicate from 1 representative of 3 independent experiments is shown. Statistical significance indicated (t test, 1-tailed distribution, P < .05). (E) Inhibitory CD4T cells were identified in the MM-BMMCs incubated in the absence or presence of lenalidomide and pomalidomide for 48 hours to 6 days. CD4T cells were gated and further analyzed for membrane CD25 and intracellular FOXP3 coexpression by flow cytometry. Top panel demonstrates CD4⁺CD25⁺FOXP3⁺ regulatory CD4T cells at the end of 6 days culture, and bottom panel represents IL-17 producing CD4 T cells (Th17) at the end of a 48-hour culture. One representative of 3 independent experiments is shown. Statistical significance indicated (t test, 1-tailed distribution, P < .05).