β-mannosylceramide activates type I natural killer t cells to induce tumor immunity without inducing long-term functional anergy

Abstract

Purpose: Most studies characterizing antitumor properties of invariant natural killer T (iNKT) cells have used the agonist, α-galactosylceramide (α-GalCer). However, α-GalCer induces strong, long-lasting anergy of iNKT cells, which could be a major detriment for clinical therapy. A novel iNKT cell agonist, β-mannosylceramide (β-ManCer), induces strong antitumor immunity through a mechanism distinct from that of α-GalCer. The objective of this study was to determine whether β-ManCer induces anergy of iNKT cells.

Experimental design: Induction of anergy was determined by ex vivo analysis of splenocytes from mice pretreated with iNKT cell agonists as well as in the CT26 lung metastasis in vivo tumor model.

Results: β-ManCer activated iNKT cells without inducing long-term anergy. The transience of anergy induction correlated with a shortened duration of PD-1 upregulation on iNKT cells activated with β-ManCer, compared with α-GalCer. Moreover, whereas mice pretreated with α-GalCer were unable to respond to a second glycolipid stimulation to induce tumor protection for up to 2 months, mice pretreated with β-ManCer were protected from tumors by a second stimulation equivalently to vehicle-treated mice.

Conclusions: The lack of long-term functional anergy induced by β-ManCer, which allows for a second dose to still give therapeutic benefit, suggests the strong potential for this iNKT cell agonist to succeed in settings where α-GalCer has failed.

Figure 1

Unlike α-GalCer, β-ManCer does not induce long-term anergy of activated iNKT cells. Mice were injected i.p. with 2.4 nmol (2 µg) of α-GalCer or β-ManCer or vehicle control. After 2 months, splenocytes were harvested and restimulated in vitro with 100 nM α-GalCer, 500 nM β-ManCer, or vehicle. (A) Proliferation was measured by ³H-thymidine uptake for the last 8 hours of a 72-hour stimulation. Stimulation index was determined by the following calculation: CPM of stimulated wells/CPM of vehicle control. (B) IFN-γ and (C) IL-4 in culture supernatants were measured after 48 hours of stimulation. (D) 1 month after injection of lipids, splenocytes were harvested and restimulated in vitro with 100 nM α-GalCer, 500 nM β-ManCer, or vehicle. TNF-α in culture supernatants was measured after 48 hours of stimulation. Representatives of at least 2 replicate experiments are shown. Bars represent mean±SD; *, statistically significantly different from vehicle control stimulation; #, statistically significant from corresponding stimulation in vehicle pretreated group p<0.05.

Figure 2

Lack of anergy induction by β-ManCer cannot be explained by selective Vβ usage of activated iNKT cells. Mice were injected with 2.4 nmol (2µg) β-ManCer or vehicle control. Splenocytes were harvested at 4 weeks, labeled with CFSE, and restimulated in vitro with 500 nM β-ManCer or vehicle for 3.5 days. At the conclusion of the restimulation, cells were harvested and analyzed by flow cytometry for Vβ expression on iNKT cells. Samples were gated on iNKT cells (defined as CD3^intermediateα-GalCer/CD1d tetramer⁺) expressing TCRβ, Vβ2, Vβ7, and Vβ8.1/8.2. The percentage of Vβ⁺ iNKT cells that had diluted CFSE was determined by analysis in FlowJo. Bars represent the percentage of Vβ⁺ iNKT cells in each group that diluted CFSE. Spleens from at least 3 mice were pooled for each group. A representative of 2 reproducible experiments is shown.

Figure 3

β-ManCer activates but does not induce sustained expression of PD-1 on iNKT cells in vivo. Mice were injected with 2.4 nmol (2 µg) α-GalCer or β-ManCer or vehicle control. At the indicated time points, splenocytes were harvested and stained for analysis by flow cytometry. Samples were gated on CD3^intermediateα-GalCer/CD1d tetramer⁺ cells and assayed for PD-1, PD-L1, CD25, and CD69 expression. (A) Representative histograms of PD-1 expression on gated iNKT cells are shown for each time point. Shaded area = control, gray line = α-GalCer, black line = β-ManCer. (B and C) Geometric mean fluorescent intensity was determined by data analysis in FlowJo for (B) PD-1 and PD-L1 and (C) CD25 and CD69 on gated iNKT cells. Representatives of at least 2 experiments are shown. Bars represent mean±SD of at least 4 mice per group. *, statistically significant vs vehicle control, p<0.05

Figure 4

β-ManCer induces less expression of PD-L1 and PD-L2 and CD86 on antigen presenting cells in vivo. Mice were injected with 2.4 nmol (2 µg) α-GalCer or β-ManCer or vehicle control. At the indicated time points, splenocytes were harvested and stained for analysis by flow cytometry. Samples were gated on (A) CD11c⁺CD11b⁻ or (B) CD11b⁺ cells and geometric mean fluorescent intensity of PD-L1, PD-L2, and CD86 was determined by data analysis in FlowJo. Representatives of at least 2 experiments are shown. Bars represent mean±SD of at least 4 mice per group. *, statistically significant vs vehicle control, p<0.05

Figure 5

iNKT cells primed by β-ManCer, but not α-GalCer, can respond to restimulation to induce tumor protection. Mice were injected with 2.4 nmol (2 µg) α-GalCer or β-ManCer or vehicle control. Two months later, CT26 cells (5 × 10⁵) were injected i.v. into the tail vein of BALB/c wild type and 50 pmol of glycolipids were administered within one hour after tumor challenge. Mice were sacrificed 14 days after tumor challenge and lung metastases were enumerated. Tumor burden was calculated by dividing the number of lung tumors in the treated groups by the mean number of tumor nodules in the respective vehicle control groups. Representatives from at least 2 independent experiments are shown. Symbols represent the tumor burden of individual mice, lines and error bars represent mean±SD. *, statistically significant vs vehicle control, p<0.05