Enhancement of ligand-dependent activation of human natural killer T cells by lenalidomide: therapeutic implications

Abstract

Natural killer T (NKT) cells are CD1d-restricted glycolipid reactive innate lymphocytes that play an important role in protection from pathogens and tumors. Pharmacologic approaches to enhance NKT cell function will facilitate specific NKT targeting in the clinic. Here we show that lenalidomide (LEN), a novel thalidomide (Thal) analog, enhances antigen-specific expansion of NKT cells in response to the NKT ligand alpha-galactosylceramide (alpha-GalCer) in both healthy donors and patients with myeloma. NKT cells activated in the presence of LEN have greater ability to secrete interferon-gamma. Antigen-dependent activation of NKT cells was greater in the presence of dexamethasone (DEX) plus LEN than with DEX alone. Therapy with LEN/Thal also led to an increase in NKT cells in vivo in patients with myeloma and del5q myelodysplastic syndrome. Together these data demonstrate that LEN and its analogues enhance CD1d-mediated presentation of glycolipid antigens and support combining these agents with NKT targeted approaches for protection against tumors.

Figure 1.

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.

Figure 2.

LEN-mediated enhancement of NKT effector function. (A) NKT cells expanded in the presence or absence of LEN, as in Figure 1A, were tested for the ability to secrete interferon-γ in response to unpulsed or α-GalCer–pulsed DCs, by intracellular cytokine secretion (ICS) assay. The right panel shows data from individual donors (n = 7). Horizontal bars represent mean percent NKT cells secreting interferon-γ. (B) TaqMan analysis showing changes in cytokine gene expression in NKT cells (expanded initially with α-GalCer–loaded DCs) and cultured overnight with unpulsed or α-GalCer–loaded DCs in the presence of absence of LEN. Data are representative of 2 separate experiments. (C) NKT cells expanded in the presence of DEX alone, or DEX + LEN, as in Figure 1D, were tested for the ability to secrete interferon-γ in response to unpulsed or α-GalCer–pulsed DCs, by ICS assay (n = 4). (D) NKT cells in freshly isolated PBMCs from healthy donors were tested for interferon-γ secretion in response to vehicle or α-GalCer–pulsed DCs, by ICS assay, as in panel A (n = 5).