We studied the function of antitumor T and natural killer T (NKT) cells from the blood and tumor bed in 23 patients with premalignant gammopathy, nonprogressive myeloma, or progressive multiple myeloma. We show that antitumor killer T cells can be detected in patients with both progressive or nonprogressive myeloma. Vα24+Vβ11+ invariant NKT cells are detectable in the blood and tumor bed of all cohorts. However, freshly isolated NKT cells from both the blood and tumor bed of patients with progressive disease, but not nonprogressive myeloma or premalignant gammopathy, have a marked deficiency of ligand-dependent interferon-γ production. This functional defect can be overcome in vitro using dendritic cells pulsed with the NKT ligand, α-galactosylceramide (α-GalCer). Fresh myeloma cells express CD1d, and can be efficiently killed by autologous NKT cells. We hypothesize that presentation of tumor derived glycolipids by myeloma cells leads to NKT dysfunction in vivo. These data demonstrate that clinical progression in patients with monoclonal gammopathies is associated with an acquired but potentially reversible defect in NKT cell function and support the possibility that these innate lymphocytes play a role in controlling the malignant growth of this incurable B cell tumor in patients.

Natural killer T (NKT) cells are distinct glycolipid reactive innate lymphocytes that are implicated in the resistance to pathogens and tumors. Earlier attempts to mobilize NKT cells, specifically, in vivo in humans met with limited success. Here, we evaluated intravenous injection of monocyte-derived mature DCs that were loaded with a synthetic NKT cell ligand, α-galactosyl-ceramide (α-GalCer; KRN-7000) in five patients who had advanced cancer. Injection of α-GalCer–pulsed, but not unpulsed, dendritic cells (DCs) led to >100-fold expansion of several subsets of NKT cells in all patients; these could be detected for up to 6 mo after vaccination. NKT activation was associated with an increase in serum levels of interleukin-12 p40 and IFN-γ inducible protein-10. In addition, there was an increase in memory CD8+ T cells specific for cytomegalovirus in vivo in response to α-GalCer–loaded DCs, but not unpulsed DCs. These data demonstrate the feasibility of sustained expansion of NKT cells in vivo in humans, including patients who have advanced cancer, and suggest that NKT activation might help to boost adaptive T cell immunity in vivo.

IntroductionNatural killer T (NKT) cells are T lymphocytes bearing NK markers that recognize glycolipid ligands in the context of CD1d molecules. 1 NKT cells play an important role in immune regulation and protection against tumors and pathogens. 1,2 These cells mediate antitumor effects by several mechanisms, including direct effects on tumor cells, strong cytokine production, antiangiogenesis, and activation of other cells, particularly NK cells and dendritic cells. 2 Thalidomide and its analog lenalidomide have shown clinical activity in myeloma and myelodysplastic syndrome (MDS). 3,4 However, the mechanism of their observed antitumor effects remains unclear. 5 Haslett et al 6 demonstrated that thalidomide can costimulate human T cells, which led to the development of lenalidomide (LEN) as an immune-modulatory derivative. 5,7,8 Initial studies of thalidomide in myeloma described an effect on NK cells. 9 However, subsequent studies have suggested that the observed effects on NK cells were indirect. 10 We hypothesized that innate CD1d-restricted NKT cells may be a proximal cellular target of LEN. Here we show that LEN greatly enhances liganddependent activation, proliferation, and cytokine production by human NKT cells, as well as enhance NKT cells in vivo. Study design Cells and materialsPeripheral blood mononuclear cells (PBMCs) from healthy donors were isolated from buffy coats purchased from New York Blood Center, or from patients with multiple myeloma, following informed consent approved by the Rockefeller University (New York, NY), Memorial Sloan-Kettering Cancer Center (New York, NY), and St Vincent's Cancer Center (New York, NY) institutional review boards. Lenalidomide (LEN; Celgene, Warren, NJ) was dissolved in dimethyl sulfoxide (DMSO; Sigma, St Louis, MO) at 1 mM. NKT ligand ␣-galactosyl-ceramide (␣-GalCer) was kindly provided by Kirin Breweries, Tokyo, Japan. Dendritic cell-mediated NKT expansionCD14 ϩ monocytes were isolated from PBMCs using immunomagnetic bead selection with CD14 microbeads and cultured in the presence of granulocyte macrophage-colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) to generate dendritic cells (DCs), as described. 11,12 After day 5 to 6 of culture, DCs were matured by adding an inflammatory cytokine cocktail as described. 12 To stimulate NKT cells, DCs were pulsed with ␣-GalCer (100 ng/mL) and cultured with CD14 Ϫ cells at a DC-to-responder ratio of 1:10 to 1:20 with or without LEN (Celgene) at 1 M, or as otherwise indicated. 12,13 For some experiments, DEX (0.5 nM) was also added as indicated at the beginning of expansion. NKT expansion was moninored by flow cytometry based on the expression of invariant T-cell receptor (V␣24/V␤11) and/or binding to CD1d-dimer loaded with ␣-GalCer, as described. 12 Intracellular cytokine staining (ICS)The ability of NKT cells to secrete cytokines following stimulation with NKT ligand (␣-GalCer) was tested using both intracellular cytokine staining and TaqMan assays, as described. 12,14 Freshly isolated PBMCs were stimulated ...

B lymphocyte-induced maturation protein-1 (BLIMP-1 or PRDI-BF1) is induced when bone marrow-derived progenitors differentiate in response to macrophage-colony stimulating factor (M-CSF) and is present in peripheral blood monocytes and granulocytes. BLIMP-1 is also induced during differentiation of U937 and HL-60 cells into macrophages or granulocytes. Induction of BLIMP-1 mRNA during macrophage differentiation of U937 and HL-60 shows a biphasic pattern. Overexpression of BLIMP-1 is sufficient to initiate macrophage differentiation of U937 cells whereas blocking endogenous BLIMP-1 inhibits differentiation. One target of BLIMP-1-dependent transcriptional repression in U937 cells is c-myc, providing an explanation for cessation of cell division. Thus BLIMP-1 is a key regulator of terminal differentiation in two separate hematopoietic lineages: myeloid cells and B lymphocytes.