Purpose: Effective prevention of graft-versus-host disease (GvHD) is a major challenge to improve the safety of allogeneic stem cell transplantation for leukemia treatment. In murine transplantation models, administration of naturally occurring CD4 + CD25+ regulatoryTcells (Treg) can prevent GvHD. Toward understanding the role of human Treg in stem cell transplantation, we studied their capacity to modulate T-cell^dependent xenogeneic (x)-GvHD in a new model where x-GvHD is induced in RAG2 administration of human peripheral blood mononuclear cells (PBMC).Experimental Design: Human PBMC, depleted of or supplemented with autologous CD25 + Tregs, were administered in mice at different doses. The development of x-GvHD, in vivo expansion of humanTcells, and secretion of human cytokines were monitored at weekly intervals. Results: Depletion of CD25 + cells from human PBMC significantly exacerbated x-GvHD and accelerated its lethality. In contrast, coadministration of Treg-enriched CD25 + cell fractions with autologous PBMC significantly reduced the lethality of x-GvHD. Treg administration significantly inhibited the explosive expansion of effector CD4 + and CD8 + Tcells. Interestingly, protection from x-GvHD after Treg administration was associated with a significant increase in plasma levels of interleukin-10 and IFN-g, suggesting the de novo development of TR1cells. Conclusions:These results show, for the first time, the potent in vivo capacity of naturally occurring humanTregs to control GvHD-inducing autologousT cells, and indicate that this xenogeneic in vivo model may provide a suitable platform to further explore the in vivo mechanisms of T-cell down-regulation by naturally occurring humanTregs.

The forkhead/winged helix transcription factor (Foxp3) is expressed as two different isoforms in humans: the full-length isoform (Foxp3FL) and an alternative-splicing product lacking the exon 2 (Foxp3DE2). We here studied the cellular distribution of Foxp3 isoforms by quantitative PCR and evaluated the functional outcome of retroviral transduction of Foxp3FL and Foxp3DE2 genes into CD4 + CD25 -cells. In PBMC, both isoforms were preferentially expressed in CD4 + CD25 hi cells. In single-cell-sorted and expanded Treg, both Foxp3 isoforms were expressed simultaneously but without a fixed ratio. Forced expression of Foxp3FL or Foxp3DE2 genes in CD4 + CD25 -T cells induced bona fide Treg that not only displayed Treg phenotype but also were anergic and mediated significant suppressive activity against CD3-activated CD4 + CD25 -cells. GFP -nontransduced cells or cells transduced with an empty vector showed no Treg phenotype, anergy or suppressive activities. In conclusion, our results reveal that both Foxp3 isoforms possess similar capacities to induce Treg; however, unnaturally high expression levels are required to convey Treg functions to CD4 + CD25 -cells. As both Foxp3 isoforms appear to be expressed in an independent fashion, studies aiming at quantification of Treg in peripheral blood or in tissue samples can benefit from determination of total Foxp3 levels rather than one of the isoforms.

Purpose: Cellular immunotherapy frequently fails to induce sustained remissions in patients with multiple myeloma, indicating the ability of multiple myeloma cells to evade cellular immunity. Toward a better understanding and effective therapeutic modulation of multiple myeloma immune evasion mechanisms, we here investigated the role of the tumor microenvironment in rendering multiple myeloma cells resistant to the cytotoxic machinery of T cells.Experimental Design: Using a compartment-specific, bioluminescence imaging-based assay system, we measured the lysis of luciferase-transduced multiple myeloma cells by CD4 þ or CD8 þ CTLs in the presence versus absence of adherent accessory cells of the bone marrow microenvironment. We simultaneously determined the level of CTL activation by measuring the granzyme B release in culture supernatants. Conclusion: These results reveal the cell adhesion-mediated induction of apoptosis resistance as a novel immune escape mechanism and provide a rationale to improve the efficacy of cellular therapies by pharmacologic modulation of CAM-IR.