Background: Invariant Natural killer T (iNKT) cells are innate lymphocytes that recognize glycolipid antigens that are presented by the MHC class I like molecule CD1d. Once activated with alpha-galactosylceramide (αGalCer), one of the most potent synthetic iNKT ligands, iNKT cells secrete a diverse array of pro- and anti-inflammatory cytokines and modulate immunity. We previously reported that adoptive transfer of highly purified iNKT cells from donor or 3rd-party mice prevented the development of GvHD through the expansion of CD4+CD25+FoxP3+ regulatory T (Treg) cells in a murine major mismatched BMT model. Though very small numbers of iNKT cells (5 x 104) could rescue the recipient mice from GvHD, applying this strategy in the clinic is challenging due to the small proportion of iNKT cells in the donor graft. Here, we attempted to expand donor iNKT cells by administrating αGalCer to donor animals with the goal of preventing acute GvHD. We utilized a liposomal formulation of αGalCer (αGalCer-lipo); that was found to have the potential of preventing GvHD in a recently published phase I/IIa clinical trial. Liposomes that do not contain αGalCer were used as a control (cont-lipo).
Methods: C57BL/6 (B6) donor mice were treated with αGalCer-lipo or cont-lipo via tail vein injection. Three days later, splenocytes were analyzed by flow cytometry. Recovered splenocytes were incubated 4 hr with PMA + ionomycin, and IFNγ and IL-4 production in iNKT cells was assessed. For in vivo testing, 1 x 106 conventional splenic T cells of αGalCer-lipo or cont-lipo treated mice were isolated (Tcon), and transferred into lethally (8.8-Gy) irradiated BALB/c recipient mice together with 5 x 106 T cell depleted bone marrow cells (TCD-BM).
Results: αGalCer-lipo treated donor mice exhibitied a 2-3 fold expansion of iNKT cells compared to cont-lipo treated animals (Figure A). iNKT cells in αGalCer-lipo treated mice showed higher expression levels of PD-1, ICOS, and Ki67 compared to cont-lipo treated mice. The proportion of Tregs, CD4+ and CD8+Tcon were similar between the two groups. CD8+T cells of αGalCer-lipo treated mice showed a small increase in Ki67 expression, however, other markers expressed by these cell populations were not different between the two treatment groups, suggesting that αGarCer-lipo treatment resulted in iNKT cell specific activation and expansion. The production of IFNγ and IL-4 in iNKT cells was higher in αGalCer-lipo treated mice compared to cont-lipo treated mice, indicating that the expanded iNKT cells were functional rather than exhausted. Recipient mice that received T cells from αGalCer-lipo treated donors showed less weight loss, lower GvHD scores, and improved survival than those from cont-lipo treated mice (p=0.0002; Figure B).
Conclusion: Preconditioning donors with αGalCer-lipo is a novel approach to utilize regulatory potential of donor iNKT cells for preventing GvHD.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.