Christine Vogtenhuber

IL-2 Receptor β-Dependent STAT5 Activation Is Required for the Development of Foxp3+ Regulatory T Cells

IL-2−/− mice develop autoimmunity despite having relatively normal numbers of regulatory T cells (Tregs). In contrast, we demonstrate that IL-2−/− × IL-15−/− and IL-2Rβ−/− mice have a significant decrease in Treg numbers. Ectopic expression of foxp3 in a subset of CD4+ T cells rescued Treg development and prevented autoimmunity in IL-2Rβ−/− mice, suggesting that IL-2Rβ-dependent signals regulate foxp3 expression in Tregs. Subsequent analysis of IL-2Rβ-dependent signal transduction pathways established that the transcription factor STAT5 is necessary and sufficient for Treg development. Specifically, T cell-specific deletion of STAT5 prevented Treg development; conversely, reconstitution of IL-2Rβ−/− mice with bone marrow cells expressing an IL-2Rβ mutant that exclusively activates STAT5 restored Treg development. Finally, STAT5 binds to the promoter of the foxp3 gene suggesting that IL-2Rβ-dependent STAT5 activation promotes Treg differentiation by regulating expression of foxp3.

IL-21 blockade reduces graft-versus-host disease mortality by supporting inducible T regulatory cell generation

Interleukin-21 (IL-21) enhances T helper 1 (Th1) and Th17 differentiation while inhibiting the conversion of inducible regulatory T cells (Tregs) from naive T cells. To determine the role of IL-21 in graft-versus-host disease (GVHD), anti-IL-21 antibody (Ab) was given to recipients of CD25(-)CD4(+) or CD4(+) and CD8(+) T-effectors. IL-21 neutralization attenuated GVHD-related weight loss and prolonged survival. Likewise, a majority of mice receiving IL-21(-/-) CD25(-) T-effectors survived long term, whereas those receiving wild-type T cells died. The latter recipients had higher grades of GVHD in the ileum and colon. Surprisingly, disruption of IL-21 signaling did not affect IL-17 production, although colon-infiltrating T-effector cells had decreased interferon gamma (IFNgamma) and increased IL-4 production. FoxP3(+) Tregs were increased in colons of anti-IL-21 Ab-treated recipients of FoxP3(-) IL-21(-/-) T cells, indicating Treg conversion. Recipients of FoxP3-deficient T-effectors isolated from chimeras were resistant to the GVHD protective effects of IL-21 blockade. Whereas graft-versus-leukemia (GVL) can occur in the absence of IL-21, loss of both IL-21 and perforin expression abrogated GVL. Together, these data indicate that IL-21 suppresses inducible Treg conversion and further suggest that IL-21 blockade is an attractive strategy to reduce GVHD-induced injury.

Constitutively active Stat5b in CD4+ T cells inhibits graft-versus-host disease lethality associated with increased regulatory T-cell potency and decreased T effector cell responses.

Overexpression of a constitutively active form of Stat5b (Stat5b-CA) increases regulatory T cells (Tregs). We show that Stat5b-CA transgenic (TG) CD4(+) T cells had a markedly reduced graft-versus-host disease (GVHD) capacity versus wild-type (WT) T cells. Stat5b-CA TG versus WT CD4(+) T cells had a higher proportion of Tregs, which were superior in suppressing alloresponses mediated by CD4(+)CD25(-) effector T cells (Teffs). By day 5 after transplantation, Stat5b-CA TG Tregs had expanded approximately 3-fold more than WT Tregs. Purified Stat5b-CA TG Tregs added to WT CD4(+)CD25(-) Teffs were superior on a per-cell basis for inhibiting GVHD versus WT Tregs. Surprisingly, rigorously Treg-depleted Stat5b-CA TG versus WT CD4(+)CD25(-) Teffs caused less GVHD lethality associated with diminished Teff proinflammatory and increased Th2 anti-inflammatory cytokine responses. Reduced GVHD by Stat5b-CA TG versus WT Teffs could not be explained by conversion into Tregs in day 10 posttransplantation spleen or small intestine. In addition, Stat5b-CA TG Teffs retained a graft-versus-leukemia response. These results indicate a major role for Stat5 in Treg expansion and potency along with a lesser but significant role in Teff activation and suggest a strategy of pharmacologic Stat5b up-regulation as a means of decreasing GVHD while retaining a graft-versus-leukemia effect.

Ex vivo Inhibition of NF-κB Signaling in Alloreactive T-cells Prevents Graft-versus-host Disease

The ex vivo induction of alloantigen‐specific hyporesponsiveness by costimulatory pathway blockade or exposure to immunoregulatory cytokines has been shown to inhibit proliferation, IL‐2 production, and the graft‐versus‐host disease (GVHD) capacity of adoptively transferred T‐cells. We hypothesized that inhibition of the intracellular NF‐κB pathway in alloreactive T‐cells, which is critical for T‐cell activation events including IL‐2 transcription, could lead to alloantigen hyporesponsiveness and loss of GVHD capacity. We demonstrate that treatment of mixed lymphocyte reaction (MLR) cultures with PS1145, a potent inhibitor of NF‐κB activation, can induce T‐cell hyporesponsiveness to alloantigen in primary and secondary responses while preserving in vitro responses to potent mitogenic stimulation. GVHD lethality in recipients of ex vivo PS1145‐treated cells was profoundly inhibited. Parking of control or PS1145‐treated MLR cells in syngeneic Rag−/− recipients resulted in intact contact hypersensitivity (CHS) responses. However, GVHD lethality capacity also was restored, suggesting that lymphopenic expansion uncoupled alloantigen hyporesponsiveness. These results indicate that the NF‐κB pathway is a critical regulator of alloresponses and provide a novel small molecule inhibitor based approach that is effective in preventing early posttransplant GVHD lethality but that also permits donor T‐cell responses to recover after a period of lymphopenic expansion.

Outgrowth of CD4low/negCD25+ T Cells with Suppressor Function in CD4+CD25+ T Cell Cultures upon Polyclonal Stimulation Ex Vivo

CD4+CD25+ regulatory T cells (Tregs) play an essential role in controlling autoimmunity and allograft rejection. Several ex vivo activation and expansion protocols have been developed to amplify cell numbers and suppressor function of murine and human Tregs. We demonstrate in this study that ex vivo activation and expansion of murine Tregs resulted in an enrichment of a CD4low/negCD25+ T cell population that was more than 20-fold more potent than expanded conventional Tregs in suppressing an in vitro CD4+CD25− T cell response to allo-Ag. The generation of CD4low/negCD25+ T cells was independent of the presence of Tregs in the culture, and suppressor function was acquired only after activation and expansion. CD4low/negCD25+ T cells expressed either an αβ or γδ TCR, had an activated phenotype, and did not express the transcription factor FoxP3. Despite expressing the cell surface Ags lymphocyte activation gene-3 (CD223) and CD103, neither was essential for suppressor cell function. Suppression by CD4low/negCD25+ T cells was prevented by a semipermeable membrane and was independent of IL-10 and TGF-β. In summary, we describe in this study CD4low/negCD25+ FoxP3neg T cells with highly potent suppressor cell function derived from cultures of an enriched population of CD4+CD25+ T cells that may contribute to the suppressor activity of ex vivo expanded bone fide Tregs.