Background: Although regulatory T cells (Tregs) are beneficial for transplant-tolerance induction, decisive methods for their preferential expansion have not yet been established. In our series of reports, we showed that Foxp3+CD4+CD25+ Treg expansion in an early phase after invariant natural killer T (iNKT) cell stimulation by liposomal α-galactosylceramide (lipo-αGC) with CD154 blockade was essential for establishing long-term mixed hematopoietic chimerism in an allogenic murine bone marrow transplant model. In this study, we applied our in vivo protocol to ex vivo Treg expansion using culture systems containing either murine splenocytes (mSPCs) or human peripheral blood monocular cells (hPBMCs) and identified critical cytokines in these responses.

Methods: mSPCs (2[times]105) from BALB/c mice or hPBMCs (2[times]105) from healthy volunteers were cultured in the absence or presence of lipo-αGC with or without anti-CD154 neutralizing monoclonal antibody (mAb). Ki67 expression of the Foxp3+CD25+ population CD4+ T cells was analyzed using flow cytometry 24 or 96 h after the start of the culture, in the absence or presence of neutralizing mAbs for IL-2, IL-4, or IL-10. The IFN-γ, IL-2, IL-4, or IL-10 concentration in the culture supernatants was measured using a Cytometric Bead Array.

Results: The proportion of Ki67+ Tregs increased in both hPBMC and mSPC cultures with lipo-αGC, but not in those with anti-CD154 mAb. All measured cytokines were detected in substantial amounts in the culture supernatants of both mSPCs and hPBMCs with lipo-αGC. To clarify the role of cytokines in in vitro Treg expansion, iNKT cell-deficient and wild type-derived mSPCs were separated using transwell culture dishes, and then cultured with lipo-αGC. We observed Treg expansion in the iNKT cell-deficient mSPCs. Among the various cytokines detected after the culture with lipo-αGC, we first tested the role of IL-2. We observed that Treg expansion was reduced in the culture with lipo-αGC after adding anti–IL-2 neutralizing mAb.

Conclusion: The results suggest that the IL-2 production of lipo-αGC–stimulated iNKT cells is indispensable for the ex vivo expansion of Tregs derived from hPBMCs and mSPCs.

CITATION INFORMATION: Katsumata H, Ikemiyagi M, Miyairi S, Ishii R, Kanzawa T, Fukuda H, Hirai T, Okumi M, Ishii Y, Tanabe K. A Novel Method for iNKT Cell-Mediated Ex Vivo Treg Expansion Applied to Induce Human Transplant Tolerance. Am J Transplant. 2017;17 (suppl 3).

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