Background: We previously established mixed chimerism after invariant natural killer T-cell stimulation by administration of liposomal α-galactosylceramide (lipo-αGC) and costimulatory blockade, leading to regulatory T cell (Treg) expansion in mice. In this regimen, the thymus was considered important because Tregs failed to expand in thymectomized mice, resulting in a chimerism brake. In the thymus, Tregs are known to develop through a two-step process: first, post-positive-selection thymocytes differentiate into CD4⁺CD25⁺Foxp3⁻ Treg precursors (preTregs) in a T cell receptor (TCR)-dependent manner, and second, IL-2 stimulation causes these preTregs to develop into Tregs. Herein, we investigated the effect of lipo-αGC on this process by using a primary thymocyte culture system.

Methods: Thymocytes from BALB/c mice were incubated with lipo-αGC for 3 days. Thereafter, the cell population was analyzed and levels of various cytokines (interleukin (IL)-2, IL-4, IL-10, and interferon-γ) were measured in the culture supernatant by flow cytometry using the Cytometric Beads Array. To evaluate their ability to differentiate into Foxp3+ Tregs, CD4⁺CD25⁺Foxp3⁻ cells sorted from lipo-αGC-stimulated thymocytes of Foxp3-GFP BALB/c mice were stimulated with IL-2 for 24 hours, and Foxp3-GFP expression was then analyzed by flow cytometry.

Results: CD4⁺CD25⁺Foxp3⁺ Treg and CD4⁺CD25⁺Foxp3^– cell numbers increased in lipo-αGC-stimulated thymocytes compared to that in unstimulated thymocytes. Cytokine production levels between the two groups were comparable. Thus, lipo-αGC stimulation may affect the first step[mdash]TCR-dependent preTreg expansion[mdash]in the aforementioned two-step process. Foxp3 expression in CD4⁺CD25⁺Foxp3^– cells was induced in an IL-2-dependent manner. Taken together, these results indicate that the CD4⁺CD25⁺Foxp3^– cell population contained preTregs.

Conclusions: In vitro lipo-αGC stimulation can cause preTreg expansion followed by Treg development, suggesting that invariant natural killer T cells may be involved in regulation of thymic Treg differentiation and development.

CITATION INFORMATION: Katsumata H., Ikemiyagi M., Kanzawa T., Fukuda H., Ishii R., Saiga K., Okumi M., Ishii Y., Tanabe K. In Vitro α-Galactosylceramide Stimulation Expands CD4⁺CD25⁺Foxp3⁻ Regulatory T Cell Precursors in Murine Thymocytes Am J Transplant. 2017;17 (suppl 3).

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