LEN/thalidomide-mediated enhancement of NKT cells in vitro and in vivo. (A) LEN boosts expansion of NKT cells from healthy donors by DCs pulsed with α-GalCer. T cells were expanded with DCs loaded with α-GalCer (or unpulsed DCs) in the presence or absence of LEN/DMSO control. After 2 weeks of culture, the presence of Vα24⁺Vβ11⁺ or CD1d-aGC dimer⁺ NKT cells was monitored by flow cytometry. (B) LEN boosts expansion of NKT cells by DCs pulsed with α-GalCer in both healthy donors and patients with myeloma. NKT cells were expanded with DCs loaded with α-GalCer (or unpulsed DCs) in the presence or absence of LEN/DMSO control. After 1 to 2 weeks of culture, the presence of Vα24⁺Vβ11⁺ NKT cells was monitored by flow cytometry. Data shown are fold increase in NKT cells for cultures with α-GalCer–loaded DCs. Horizontal bars represent mean NKT expansion. (C) Immature versus mature DCs. NKT cells were expanded as in panel A, with immature monocyte-derived DCs (iDCs), or after DC maturation with inflammatory cytokines (MDCs). After 2 weeks of culture, the presence of Vα24⁺Vβ11⁺ NKT cells was monitored by flow cytometry. Data are representative of 8 separate experiments. (D) Comparison of DEX versus DEX + LEN. NKT cells were expanded as in panel A, in the presence of DEX (0.5 nM) alone, or with LEN (1 μM). Data shown are percent iNKT cells. Horizontal bars represent mean NKT expansion. (E) Expansion of NKT cells in myeloma. Circulating NKT cells were monitored by flow cytometry before and 1 month after thalidomide therapy in patients with myeloma (n = 2). (F) NKT expansion in patients with MDS (n = 5) treated with LEN. iNKT cells were quantified by flow cytometry before and at the start of the third and fifth/sixth cycle of LEN therapy. Closed symbols represent patients with del5q.