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The adoptive transfer of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in murine models of allogeneic hematopoietic cell transplantation (HCT) has been shown to protect recipient mice from lethal acute graft-versus-host disease (GVHD) and this approach is being actively investigated in human clinical trials. Here, we examined the effects of cryopreservation on Tregs. We found that freeze and thaw of murine and human Tregs is associated with reduced expression of L-selectin (CD62L), which was previously established to be an important factor that contributes to the in vivo protective effects of Tregs. Frozen and thawed murine Tregs showed a reduced capacity to bind to the CD62L binding partner MADCAM1 in vitro as well as an impaired homing to secondary lymphoid organs in vivo. Upon adoptive transfer frozen and thawed Tregs failed to protect against lethal GVHD compared with fresh Tregs in a murine model of allogeneic HCT across major histocompatibility barriers. In summary, the direct administration of adoptively transferred frozen and thawed Tregs adversely affects their immunosuppressive potential which is an important factor to consider in the clinical implementation of Treg immunotherapies.

Interleukin (IL)-18 stimulates T helper 1 (Th1) and Th2-mediated immune responses, and has been shown to modulate acute graft-versus-host disease (aGVHD). It is still unknown whether increased IL-18 levels during aGVHD are of host or donor origin, and how the absence of IL-18 has an impact on migration and expansion of conventional CD4(+)CD25(-)(Tconv) and CD4(+)CD25(+) regulatory (Treg) T cells in vivo. By utilizing IL-18 gene-deficient donor versus recipient animals we found that the major cytokine production during the early phase of aGVHD induction was recipient derived, whereas donor hematopoietic cells contributed significantly less. By generating IL-18(-/-) luciferase transgenic mice we were able to investigate the impact of IL-18 on Tconv and Treg expansion and trafficking with in vivo bioluminescence imaging. Although migration to secondary lymphoid organs did not have a significantly impact from the absence of host IL-18, Tconv but not Treg expansion increased significantly. Absence of host IL-18 production translated into lower IFN-gamma levels in the early phase after transplantation. We conclude that host-derived IL-18 is a major factor for IFN-gamma production that may have a protective effect on CD4(+)-mediated aGVHD, but is nonessential for Treg expansion in an allogeneic environment.