Rapid and reliable generation of invariant natural killer T-cell lines in vitro

Abstract

Several tools have proved useful in the study of invariant natural killer T (iNKT) cells, including CD1d-deficient mice, J alpha281-deficient mice, synthetic lipid antigens and antigen-loaded CD1d tetramers. However, the generation and examination of long-term primary murine iNKT cell lines in vitro has been challenging. Here, we show the rapid generation of iNKT cell lines from splenic iNKT cells of V alpha14 T-cell receptor (TCR) transgenic (Tg) mice. These purified iNKT cells were stimulated by bone marrow-derived dendritic cells (BMDCs) loaded with alpha-galactosylceramide (alphaGalCer) and cultured with interleukin (IL)-2 and IL-7. iNKT cells proliferated dramatically, and the cell number exhibited a 100-fold increase within 2 weeks and a 10(5)-fold increase in 8 weeks after repeated stimulation with alphaGalCer. The iNKT cell lines consisted of iNKT cells expressing V beta chains including V beta8.1/8.2, V beta14, V beta10, V beta6 and V beta7, and responded to stimulation with alphaGalCer presented both by BMDCs and by plate-bound CD1d. In addition, the iNKT cell lines produced interferon (IFN)-gamma when activated by lipopolysaccharide (LPS) or CpG oligodeoxynucleotide (ODN)-stimulated BMDCs. Further, we show that iNKT cell lines produced cytokines in response to microbial antigens. In summary, high-yield iNKT cell lines were generated very rapidly and robustly expanded, and these iNKT cells responded to both TCR and cytokine stimulation in vitro. Given the desire to study primary iNKT cells for many purposes, these iNKT cell lines should provide an important tool for the study of iNKT cell subsets, antigen and TCR specificity, activation, inactivation and effector functions.

Figure 1

Rapid generation of murine invariant natural killer T (iNKT) cell lines in vitro. iNKT cells were purified from spleens of Vα14 transgenic (Tg) mice. 2 × 10⁶ iNKT cells were co-cultured with 2 × 10⁵ bone marrow dendritic cells (BMDCs) loaded with α-galactosylceramide (αGalCer) (100 ng/ml). Three to five days later, m (murine) IL-2 (10 U/ml) and mIL-7 (10 ng/ml) were added to the culture medium, and cells were stimulated with αGalCer-BMDCs every 2–3 weeks. (a) Fold increase in cell numbers of four independent cell lines (AC6, AC8, AC9 and AC10) at the indicated time-points. (b) Anti-CD3e-fluorescein isothiocyanate (FITC) and CD1d tetramer-allophycocyanin staining of an iNKT cell line at 8 weeks post in vitro culture. Results are representative of two separate experiments. (c) Vβ usage of iNKT cell lines. iNKT cell lines (AC3, AC4, AC6, AC7, AC8, AC9 and AC10) were stained with a panel of anti-Vβ monoclonal antibodies (mAbs). Frequencies of iNKT cells with indicated Vβ chains are expressed as percentages in the 100% stacked column chart. Each column represents the percentage of iNKT cells with the indicated Vβ chains contributing to the total iNKT cells.

Figure 2

Invariant natural killer T (iNKT) cell lines produce cytokines after α-galactosylceramide (αGalCer) stimulation. iNKT cell lines (AC6, AC8 and AC10) stimulated with αGalCer more than 2 weeks earlier were washed and re-suspended with complete RPMI medium without cytokines. (a) 1 × 10⁵ iNKT cells were incubated with 1 × 10⁵ bone marrow dendritic cells (BMDCs) and αGalCer (100 ng/ml). Supernatants were collected and cytokine production was measured by enzyme-linked immunosorbent assay (ELISA). (b) 1 × 10⁵ iNKT cells were incubated with 1 × 10⁵ wild-type (WT) BMDCs or CD1d^−/− BMDCs and αGalCer. The interferon (IFN)-γ content in culture supernatants was determined by ELISA. Results are representative of two separate experiments.

Figure 3

Invariant natural killer T (iNKT) cell lines become activated and secrete cytokines in response to α-galactosylceramide (αGalCer) in an APC-free system. iNKT cell lines (AC6, AC8 and AC10) stimulated with αGalCer more than 2 weeks earlier were washed and re-suspended with complete RPMI medium without cytokines. 1 × 10⁵ iNKT cells were incubated in the presence of plate-bound, recombinant CD1d-Fc fusion protein loaded with αGalCer or mock loaded with dimethyl sulphoxide (DMSO). Supernatants were collected and cytokine production was measured by enzyme-linked immunosorbent assay (ELISA). Results are representative of two separate experiments.

Figure 4

Invariant natural killer T (iNKT) cell lines express cytokine receptors and become activated via a cytokine-driven mechanism. (a) Expression of surface molecules on iNKT cells. iNKT cell lines were stained with anti-CD3e-fluorescein isothiocyanate (FITC), CD1d-tetramer-allophycocyanin, and a panel of surface molecules. Expression of CD4, CD8, DX5, NK1.1, CD25, CD122, CD212 and interleukin (IL)-18R was determined on CD3-positive CD1d-tetramer-positive NKT cells. The open histograms indicate staining with each monoclonal antibody (mAb) and the grey histograms represent the isotype controls. Results are representative of two separate experiments. (b) iNKT cell line activation by lipopolysaccharide (LPS)-stimulated bone marrow dendritic cells (BMDCs) is CD1d and MyD88 dependent. 1 × 10⁵ NKT cells were incubated with LPS (10 μg/ml) or α-galactosylceramide (αGalCer; 10 ng/ml) in the presence of 1 × 10⁵ wild-type (WT) BMDCs, CD1d^−/− BMDCs, or MyD88^−/− BMDCs. Supernatants were collected and interferon (IFN)-γ production was determined by enzyme-linked immunosorbent assay (ELISA). (c) CD1d- and MyD88-dependent iNKT cell line activation by CpG oligodeoxynucleotide (ODN). 1 × 10⁵ NKT cells were incubated with CpG ODN (125 ng/ml) in the presence of 1 × 10⁵ WT BMDCs, CD1d^−/− BMDCs or MyD88^−/− BMDCs. IFN-γ production in supernatants was measured by ELISA. Results are representative of two separate experiments.

Figure 5

Invariant natural killer T (iNKT) cell lines respond to a microbial antigen and produce cytokines. 1 × 10⁵ NKT cells were incubated with the indicated concentrations of glycosphingolipid (GSL)-1 or α-galactosylceramide (αGalCer; 10 ng/ml) in the presence of 1 × 10⁵ wild-type (WT) BMDCs or CD1d^−/− BMDCs. Supernatants were collected to determine interferon (IFN)-γ and interleukin (IL)-4 production by enzyme-linked immunosorbent assay (ELISA). Results are representative of two separate experiments.