Mouse and human iNKT cell agonist β-mannosylceramide reveals a distinct mechanism of tumor immunity
Jessica J. O’Konek, Petr Illarionov, Deborah Stewart Khursigara, Elena Ambrosino, Liat Izhak, Bernard F. Castillo II, Ravinder Raju, Maryam Khalili, Hee-Yong Kim, Amy R. Howell, Gurdyal S. Besra, Steven A. Porcelli, Jay A. Berzofsky, Masaki Terabe
Jessica J. O’Konek, Petr Illarionov, Deborah Stewart Khursigara, Elena Ambrosino, Liat Izhak, Bernard F. Castillo II, Ravinder Raju, Maryam Khalili, Hee-Yong Kim, Amy R. Howell, Gurdyal S. Besra, Steven A. Porcelli, Jay A. Berzofsky, Masaki Terabe
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Research Article Immunology

Mouse and human iNKT cell agonist β-mannosylceramide reveals a distinct mechanism of tumor immunity

Abstract

Type 1 or invariant NKT (iNKT) cell agonists, epitomized by α-galactosylceramide, protect against cancer largely by IFN-γ–dependent mechanisms. Here we describe what we believe to be a novel IFN-γ–independent mechanism induced by β-mannosylceramide, which also defines a potentially new class of iNKT cell agonist, with an unusual β-linked sugar. Like α-galactosylceramide, β-mannosylceramide directly activates iNKT cells from both mice and humans. In contrast to α-galactosylceramide, protection by β-mannosylceramide was completely dependent on NOS and TNF-α, neither of which was required to achieve protection with α-galactosylceramide. Moreover, at doses too low for either alone to protect, β-mannosylceramide synergized with α-galactosylceramide to protect mice against tumors. These results suggest that treatment with β-mannosylceramide provides a distinct mechanism of tumor protection that may allow efficacy where other agonists have failed. Furthermore, the ability of β-mannosylceramide to synergize with α-galactosylceramide suggests treatment with this class of iNKT agonist may provide protection against tumors in humans.

Authors

Jessica J. O’Konek, Petr Illarionov, Deborah Stewart Khursigara, Elena Ambrosino, Liat Izhak, Bernard F. Castillo II, Ravinder Raju, Maryam Khalili, Hee-Yong Kim, Amy R. Howell, Gurdyal S. Besra, Steven A. Porcelli, Jay A. Berzofsky, Masaki Terabe

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Figure 3

β-ManCer activates mouse iNKT cells.

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β-ManCer activates mouse iNKT cells. (A) Mouse splenocytes were stimulat...
(A) Mouse splenocytes were stimulated with vehicle, α-GalCer, or β-ManCer for 18 hours, and activation was measured by FACS analysis. iNKT cells were identified as CD3intermediatePBS57/CD1d tetramer+, and CD69 and CD25 expression was determined on the gated cells. Numbers in plots represent the percentage of cells in gated population. (B) Mouse splenocytes were labeled with CFSE and stimulated with vehicle, α-GalCer, or β-ManCer for 3.5 days in the presence of anti-CD1d antibody 20H2 (10 μg/ml) or rat IgG control. Proliferation of iNKT cells, gated as in A on CD1d-tetramer+ and CD3-intermediate cells, was measured by CFSE dilution. (C) The proliferation of iNKT cells positive for different Vβ chains was measured by CFSE dilution. % Divided, percentage of Vβ+ CD1d tetramer+ iNKT cells which divided. (D) 24.9.E and DN32.D3 hybridoma cells were stimulated with α-GalCer–loaded (black squares) or β-ManCer–loaded (white squares) mCD1d dimers for 24 hours. IL-2 in the supernatants was determined by ELISA. Each point represents mean ± SD of triplicates, although the SDs are too small for error bars to be visible relative to the size of the symbols. Representatives of at least 2 independent experiments are shown. (E) Liver lymphocytes were stained with α-GalCer– or β-ManCer–loaded or unloaded mCD1d dimers. The top panels illustrate staining of liver lymphocytes from WT mice, and the bottom panels illustrate liver lymphocytes from Jα18–/– mice. Representative plots from 6 experiments with reproducible results are shown. Numbers in plots represent the percentage of cells in gated population